Control of black dot in potatoes

Transcription

1 Control of lck dot in pottoes Dr. Trevor Wicks SA Reserch & Development Institute Project Numer: PT11

2 PT11 This report is pulished y Horticulture Austrli Ltd to pss on informtion concerning horticulturl reserch nd development undertken for the potto industry. The reserch contined in this report ws funded y Horticulture Austrli Ltd with the finncil support of the potto unprocessed nd vlue-dded industries. All expressions of opinion re not to e regrded s expressing the opinion of Horticulture Austrli Ltd or ny uthority of the Austrlin Government. The Compny nd the Austrlin Government ccept no responsiility for ny of the opinions or the ccurcy of the informtion contined in this report nd reders should rely upon their own enquiries in mking decisions concerning their own interests. ISBN Pulished nd distriuted y: Horticulturl Austrli Ltd Level 1 5 Crrington Street Sydney NSW 2 Telephone: (2) Fx: (2) E-Mil: horticulture@horticulture.com.u Copyright 25

3 Control of Blck dot in pottoes FINAL REPORT Horticulturl Reserch nd Development Corportion Project PT11 By Roin Hrding, T. Wicks nd B. Hll South Austrlin Reserch nd Development Institute

4 6

5 HORTICULTURAL RESEARCH AND DEVELOPMENT CORPORATION Finl Report Septemer 24 Project Title: HAL Project No.: Reserch Orgnistion: Principl Investigtor: Control of Blck dot in Pottoes PT11 South Austrlin Reserch & Development Institute GPO Box 397 Adelide South Austrli 51 Contct: Trevor Wicks Phone: Fx: Emil: wicks.trevor@sugov.s.gov.u Roin Hrding Phone: Fx: Emil hrding.roin@sugov.s.gov.u South Austrlin Reserch & Development Institute Swmp Rod Lenswood South Austrli 524 7

6 HAL Project: PT11 Dr T.J. Wicks South Austrlin Reserch nd Development Institute GPO Box 397 ADELAIDE SA 51 AUSTRALIA ph: emil: wicks.trevor@sugov.s.gov.u Blck dot, cused y the fungus Colletotrichum coccodes is mjor potto disese tht ffects oth the fresh nd processing industries. It cuses premture plnt deth, reduced yields nd downgrding of produce due to lemishes. The min im of the project is to develop mngement strtegies to help wshed potto growers nd seed growers control lck dot disese. We cknowledge the funding provided y the ntionl Potto R & D levy nd the Commonwelth Government through HAL Disclimer: Any recommendtions contined in this puliction do not necessrily represent current Horticulture Austrli policy. No person should ct on the sis of the contents of this puliction, whether s to mtters of fct or opinion or other content, without first otining specific, independent professionl dvice of the mtters set out in this puliction. 8

7 CONTENTS MEDIA SUMMARY...3 TECHNICAL SUMMARY...4 LITERATURE REVIEW...6 TECHNICAL REPORT...9 Introduction...9 Generl mterils nd methods...9 Pthogen morphology nd ecology...11 Introduction:...11 Fungl vrition...11 Effects of temperture...14 Disese development...17 Tuer infection from conidi...19 Pthogenicity of C. circnnis...21 Weeds...22 Pthogen nd plnt interction...24 Introduction:...24 Generl mterils nd methods:...24 Tuer infection points...25 Light on C. coccodes seed...28 Systemic infection...29 Systemic crry over nd disese trnsfer through soil Potto cultivrs...33 Inoculum levels...35 Wter nd hulm disese development Reglone/Dye...39 Irrigtion...4 Pivot vritions...41 MANAGEMENT...42 Introduction:...42 Generl mterils nd methods:...42 Biofumigtion...44 Inhiition from voltiles relesed from Brssic sp...44 Hydrtion effects on voltiles relesed from B. junce...46 Efficcy of voltiles relesed from B. junce seed mel in soil...47 Chemicl control...48 Invitro ssessment of tuer seed tretments...48 Effect of fungicides on trnsmission in storge...52 Effect of fungicides on crry over of seed Greenhouse screening of fungicides...55 Field nd greenhouse evlution of seed nd soil tretments...58 Evlution of fumigtion...68 Hulm desicction...77 TECHNOLOGY TRANSFER...83 RECOMMENDATIONS SCIENTIFIC AND INDUSTRY...85 ACKNOWLEDGMENTS...87 BIBLIOGRAPHY AND LITERATURE CITED...89 APPENDICES

8 2

9 MEDIA SUMMARY Over the pst 2 yers the fungl disese lck dot cused y the fungus Colletotrichum coccodes hs een reported throughout the min potto production res worldwide. For mny of these yers, growers nd reserchers hve considered it to e secondry disese of low importnce. However incresing demnds on tuer qulity in oth the fresh nd processing industries hve shown it to e n economiclly importnt disese due to externl skin lemishes, internl tissue discolourtion of stem ends nd yield reductions. Austrlin, USA nd Isreli trils hve shown lck dot reduced yields y etween 12-3%. The min im of this project ws to develop mngement strtegies to control the disese for oth wshed potto growers nd seed growers. Tril results hve shown tht there is high incidence of lck dot oth on nd in seed pottoes nd tht infected seed is the primry mens of introducing the disese into new regions. One tril where clen seed ws plnted into new ground, etween 15 nd 88% of the dughter tuers were infected. Seed tretments with the fungicides Mxim nd Amistr were the most effective fungicides t reducing the impct of this disese ut only where soil levels of the fungus were low. Where soil levels were high, fumigtion with Methm, Telone II or in-furrow tretments of Amistr reduced the impct of the disese. However if infected seed is plnted under these conditions, disese incidence nd severity cn e higher thn where no soil tretment ws pplied. A secondry infection cycle ws shown to occur etween crop mturity/hulm desicction nd hrvest. Folir ppliction of Amistr or withholding irrigtion efore hrvest reduced the incidence of the disese. Less effective were the ppliction of chemicls nd slshing/removl of hulm tissue. Further studies re needed to determine optimum timing of these tretments nd dose of fungicides. This will ensure tht growers hve diversity of control methods nd not rely solely on chemicl methods tht could give rise to fungicide resistnt strins of fungi. Becuse lck dot survives in the soil for up to 8ys, it is recommended tht rottions of t lest this length e used. During this time non-host crops should e used nd weed species such s ft hen, skeleton weed nd lck nightshde tht re lso hosts should e controlled. Plnting or hving crops mturing when the soil tempertures re ove 24 o C increses disese incidence. Therefore growers should void these periods nd if crops do mture during this time, lifting tuers erlier will reduce disese levels. 3

10 TECHNICAL SUMMARY Blck dot cused y the fungus C. coccodes is now recognised s mjor disese of pottoes worldwide. Lortory, glsshouse nd field trils hve een conducted in South Austrli since 21 to develop mngement strtegies to control the disese. The mjor findings for these studies were: 1. Lortory nd glsshouse studies showed tht the fungus ws most ctive round 24 o C nd cused most disese round this temperture. 2. Disese development ws monitored in nturlly infected plnts, which showed tht the symptoms developed on the stem round 74 dys fter plnting wheres tuer infection ws detected 1 dys lter. 3. Levels of C. coccodes were mesured on potto tuer seed used in South Austrli. In 21, 61% of seed lots were infected with lck dot nd 93% in 22. In some smples ll tuers were infected with C. coccodes. Vsculr infection (internl) levels s high s 3% were lso found in some smples. 4. Glsshouse studies showed tht dughter seed could e infected s result of systemic growth of the fungus from the infected mother seed. However seed tuers with externl skin infection produced dughter tuers with the highest incidence nd severity of C. coccodes. 5. Glsshouse studies were mde to determine the effect of different soil inoculum levels on the development of C. coccodes. Plnts grown in 2 microscleroti/gm of soil senesced more rpidly thn those in 1 or 15 microscleroti/gm ut there ws no difference in the numers of tuers infected or the severity of infection on tuers produced in the different inoculum levels. 6. Fourteen potto cultivrs were evluted for susceptiility to C. coccodes in nturlly infected soil. Differences in incidence of stem coloniztion nd severity of tuer infection were oserved etween different cultivrs, ut none were immune. Desiree ws the lest infected wheres Colin ws the most susceptile of the 14 cultivrs evluted. 7. Weed hosts were collected from potto fields etween nd exmined for presence of C. coccodes on stems nd roots. The fungus ws detected on common weeds such s ft hen (Chenopodium llium), Blck night Shde (Solnum nigrum), Heliotrope (Heliotropium europium), Skeleton weed (Chondrill junce) nd others. 8. Overhed irrigtion pplied to infected hulms just prior to hrvest incresed the incidence nd severity of tuer infection compred to those not irrigted Experiments showed tht conidi wshed from infected stems or pplied to the soil surfce re cple of infecting tuers eneth the soil. Experiments with fluorescent dye indicted tht wter from overhed irrigtion wshes spores down the edge of the stem nd long the stolon nd roots to the tuer. Exmintion of C. coccodes infected tuers cross rdius of pivot showed tht the highest levels of incidence nd severity were t the centre of the pivot where most wter is pplied nd lowest t the extremity where lest wter is pplied. 9. The ppliction of the desiccnt Reglone prior to hrvest did not increse the level of C. coccodes infection. However the ppliction of the fungicide Amistr one week fter Reglone reduced oth the incidence nd severity of C. coccodes on tuers in some experiments. Other experiments confirmed the oservtion tht the longer tuers remined in the soil fter hulm destruction the more disese develops. 4

11 1. Severl experiments were crried out to evlute the potentil of iofumignts from Brssic s s mens of controlling C. coccodes. Leves, roots nd mel from B. Junce, B. npus nd R stivus were evluted in-vitro for their inhiitory effects on mycelil growth of C. coccodes. The results showed tht mel of B. Junce ws most inhiitory, ut there were vritions etween Brssic species nd plnt prts. Further experiments were crried out evluting the efficcy of voltiles relesed from B. junce mel in soil nd n inhiiting effect ws demonstrted. 11. Mny fungicides nd rtes were evluted in lortory, glsshouse nd field trils s tuer seed tretments for the control of C. coccodes. Of the 12 fungicides tested Mxim, Amistr, Crio nd Octve were the most effective. 12. Lrge scle field trils were lso undertken to evlute the efficcy of soil fumigtion (Methm nd Telone ) for the control of C. coccodes. These trils lso included the evlution of fungicide tuer seed tretments nd the use of Amistr. The effects of soil fumigtion were vrile nd were compromised if used in conjunction with infected tuers 5

12 LITERATURE REVIEW Distriution of the disese. Blck dot, disese of pottoes cused y the fungus Colletotrichum coccodes (Wllr.) hs een reported throughout the world (4, 24, 36, 53, 63) including Chile (23), Europe (2, 58), Austrli (26), South Afric (15), nd Isrel (67). Blck dot hs een reported s minor disese (58, 6, 63), however it is now recognized s mjor pthogen of pottoes, cusing sustntil economic losses in mny countries (2, 4, 15, 16, 33, 58, 66). Hosts Overses studies hve shown tht it hs wide host rnge tht includes t lest 58 species nd 17 fmilies, primrily vegetles within solncee (55). In ddition there re numer of hosts tht re clssified s weeds (see ppendix) (55). These include severl solnceous weed species; velvetlef nd estern lck nightshde (1, 55, 74). Unmnged weed popultions could result in the uild up of inoculum levels etween rottions or my ct s source of primry inoculum for susequent potto crops. Identifying the host plnts in Austrli should llow growers to develop etter disese mngement prctices through selective weed control nd non-host rottionl crops. The cusl pthogen Overses studies showed tht there is lrge degree of vriility in sclerotium size, colour, eril mycelium nd virulence mong isoltes of C. coccodes (4, 4, 67). For exmple some isoltes do not produce scleroti (4). Vritions mong Austrlin isoltes hve not een studied nd could show tht yield losses due to C. coccodes infection occur without the development of scleroti. Effect on yield Studies in the USA hve shown tht on poorly drined, sndy soils, C. coccodes cuses significnt yield losses (4, 33). Yield losses hve lso een recorded in Isrel, UK nd Austrli (42, 65) nd vry etween different cultivrs in oth Isrel nd the UK (57, 66, 68). Whilst yield losses hve gone mostly unrecognised in Austrli, growers hve oserved mrked reduction in qulity nd pckers involved in the production of wshed pottoes. In n ttempt to reduce the decline in qulity some growers hve moved production to new ground only to find similr levels of infection. Disese epidemiology In South Austrli recent studies hve shown tht lck dot is significnt prolem on seed tuers, with 1% infection in some smples of certified seed tuers (14, 25). In ddition the lck dot fungus hs een recovered internlly from tuers in 75% of 54 seed lots tested (25). Similr levels of lck dot on seed tuers hve lso een reported overses (16, 33, 37). The effect the tuer seed infection hs on the qulity of dughter tuers nd disese crry over hs not een studied extensively. In soil the fungus my exist s either scleroti or conidi t undetectle levels. Scleroti survive in the soil for up to 8 yrs (18), nd while the conidi cn survive for up to 1 yr in soil only few survive longer thn few weeks (7, 22). Scleroti survived longer nd more consistently when they were free in the soil rther thn ssocited with plnt tissue (19). Recent overses reports lso show C. coccodes to e n ir-orne pthogen (4, 34, 46) cusing folir infection (13), however it is not known how long conidi survive on folige. 6

13 Studies hve lso shown tht the incidence nd severity of lck dot increses on unwshed tuers when stored t 15 o C ut not t 5 o C (56) nd tht hrvesting erly nd storing tuers dry my prevent or reduce disese expression (48). C. coccodes cn lso exist in synergistic reltionship with one or more other pthogens such s V. dhlie (36, 53). Environmentl fctors tht influence disese expression Blck dot is most frequently ssocited with light sndy soils, low nitrogen, high tempertures, nd poor wter dringe (69). However ltent infections in stressed plnts mke the effect of the iotic nd iotic fctors on disese development difficult to determine. In the USA, excessive rin, irrigtion nd low tempertures erly in the seson followed y prolonged drought resulted in disese expression (63). Whilst in Englnd irrigtion reduced stem, root nd tuer infection up to 18 weeks fter plnting, ut lter rotting of tuers ws incresed y irrigtion (56). In Isrel where the crop is irrigted routinely, disese incidence nd yield loss were oserved when tempertures were high nd soil reltively dry (69). Growth chmer studies showed tht short dy lengths (8:16 L: D) rther thn longer dy lengths resulted in higher incidence nd severity of lck dot on tuers (68). Control Chemicl: A numer of chemicls hve een evluted s seed tretments to reduce dughter tuer infection (2, 27, 43, 7), ut few studies hve een reported on new fungicides with different modes of ction. One group, the stroilurins, re prticulrly effective on Colletotrichum sp. nd evidence from the USA indictes tht the use of these fungicides my e mjor rekthrough in the control of lck dot (34). Evluting these new fungicides for use s seed tretments, folir tretments or infurrow soil tretments my provide new strtegies for the control of lck dot. Since seed tretments re unlikely to control tuer infection where the levels of soil inoculum re high, soil solriztion or fumigtion my reduce soil levels of the pthogen. However in the USA nd Isrel fumigtion with methyl romide, vpm or formlin sometimes filed to control C. coccodes (63, 68). Breeding: Breeding less susceptile cultivrs is nother mens of controlling C. coccodes (22, 4, 68, 69) ut no studies on susceptiility of cultivrs hve een crried out in Austrli. In the UK the commercil potto cultivrs Desiree, Mris Piper, Mris Peer nd Record showed significntly higher infection levels whilst Cr, Pentlnd Crown nd Romno showed the lest infection (57). In Isrel the cultivrs Alph, Desiree nd Agri were more susceptile thn Cr nd Nicol (65). Culturl: Since soil levels of the fungus, environment, nd vrietl resistnce influence the timing of infection (22, 4, 65). Smpling the crop during the growing seson to determine the level of infection hs een evluted s mens to predict the pproprite hrvest time of different crops to minimize tuer infection (41). Results showed tht whilst this method of disese prediction worked with one cv. Estim, it ws not effective with the cv.s Mris Piper, King Edwrd or Sxon. Crop Rottions: Most growers prctise crop rottion, yet C. coccodes still cuses mjor disese prolems, even fter long reks etween potto crops. Possile resons re tht the rottions consist of other host crops (55) or the rottion intervls re to short. C. coccodes hs een shown to survive for up to 8 yers. Trils conducted in the UK found no differences on disese incidence when pottoes were grown on 2, 4 or 6 yer rottions with spring rley (28). In ddition the pthogen my e reintroduced on potto seed. Shorter crop intervls my e possile when comined with other culturl prctices such s solriztion/trping, nd mouldord ploughing (41). 7

14 Biofumigtion: Brekdown products relesed from some plnts within the Brssiccee fmily reduce fungl growth of Rhizoctoni, Pythium nd Fusrium in vitro (3) nd in field trils (11, 47) when grown or incorported in the soil. These effects re ttriuted to glucosinoltes (GSL's) relesed from Brssic residues into the soil nd eing hydrolysed y endogenous myrosinse (3). One of these rekdown products is the voltile compound isothiocynte (ITC), which is highly iocidl to nemtodes, cteri, fungi, insects nd germinting seeds (8, 54, 72). These studies suggest tht iofumignt crops hve potentil for reducing the severity of the potto disese C. coccodes. Plnting Time: Plnting dtes hve een shown to ffect the incidence of C. coccodes on pottoes (49, 65). Both studies showed reduced disese incidence on potto tuers when plnted t prticulr periods during the yer. However the results of one seson were vrile etween different yers. This my e in prt due to differences in environmentl fctors such s temperture nd rinfll from yer to yer (63). Hulm Destruction: The destruction of potto plnts is primrily used to control tuer size nd provide esy hrvest conditions. However since the development of C. coccodes coincides with crop mturity/hulm senescence, it hs een speculted tht hulm destruction my lso ply role in disese reduction. Field trils in the UK utilizing chemicl destruction with root cutting showed lower incidence of lck scurf cused y Rhizoctoni solni (55), ut the effect on lck dot hs not een investigted 8

15 TECHNICAL REPORT Introduction Blck dot cused y the fungus C. coccodes is widespred disese of pottoes in Austrli. The fungus is common in soils of potto growing res nd on seed tuers. For exmple in some certified seed, 1% of the tuers were infected with C. coccodes. Becuse the disese is so common nd develops lte in the seson, the significnce of the disese hs not lwys een recognised. Recent work from the USA showed tht C. coccodes reduces yield y more thn 5 tonnes/h (4, 34, 46). Similr yield reductions in Austrli cn e expected, ut further losses occur with wshed pottoes where infected tuers re rejected ecuse of the skin lemishes cused y the fungus. Previous work on this prolem in Austrli (42) showed tht C. coccodes reduced yields y 12% due to plnts dying premturely. Reductions t this level my e costing the industry over $1 million nnully. Additionl losses due to lemished tuers my lso e costing the South Austrlin wshed potto industry over $1.2 million per yer. Overll the high incidence of C. coccodes in seed pottoes nd the detrimentl effect on potto qulity, now mke lck dot one of the most importnt fungl diseses of pottoes in Austrli. This project involved lortory, greenhouse nd field experiments to otin n understnding of the disese nd to develop mngement strtegies to control or reduce the disese. Generl mterils nd methods Cultures of C. coccodes: - Selective medi: Two selective medi (21, 62) were evluted for their suitility to isolte C. coccodes from soil nd plnt smples. Sorrenson s NP1 medi (62) ws the most efficient t selective isoltion of C. coccodes. Isoltes: 4 isoltes of C. coccodes were otined from nturlly infected soil, potto plnts nd weeds within South Austrli, New South Wles, Victori, Tsmni nd Western Austrli. These were mintined on NP1 selective medi. Three isoltes from SA (77, 58, 17) with different morphologies were selected to use s inoculum sources for experiments, one from soil nd two from potto tuers. In ddition scleroti tht formed on the pltes were hrvested ccording to previously descried procedures (22), ir-dried nd stored t 4 o C until needed. Storge of cultures: Cultures were mintined on NP1 selective medi t 4 o C, nd sucultures of ech isolte stored t 2 o C. Detection of C. coccodes: - Plnt tissue: Stem tissue (2-5cm ove soil line) ws surfce sterilized with 1% NCL for 6 sec nd rinsed in sterile wter. Wfer thin sections of stem were mde with sterile sclpel lde nd 5 sections plted onto petri dish contining NP1 selective medi. Sp ws extrcted from the remining sl portion of ech stem section y plcing in polyethylene g nd crushing the tissue with ruer hmmer. A.5 ml liquot of sp ws pipetted onto NP1 selective medi nd seled with prfilm. After 14 dys incution t 24 o C in the drk, pltes were exmined microscopiclly nd scored for presence or sence of C. coccodes microscleroti nd the % of plnts with stem infection ws clculted. 9

16 Tuers: The stem end of tuers ws sliced nd where stining ws ovious in the vsculr tissue, 2 pieces of pprox 1mm 2 were removed with sclpel nd plted on to selective medi. After 14 dys incution t 24 o C in the drk, pltes were exmined microscopiclly nd scored for presence of C. coccodes. Assessments: - Fungl vrition: Size nd numer/cm 2 of scleroti, shpe nd presence of sete nd size of conidi were recorded 1 dys fter plting the isoltes onto NP1 selective medi nd incuted in the drk t 24 o C. Microsclerotil density nd size: A 1cm 2 grid ws plced underneth ech petri plte nd the scleroti numers counted in five rndom squres nd mens clculted. The percentge inhiition of microsclerotil density ws determined reltive to the numer of scleroti of the control. 1 scleroti were rndomly selected t 2mm rdius from the initil inoculum source nd their dimeter mesured using n oculr grticule t 4X mgnifiction. Mycelil growth: Fungl growth ws mesured s the men of two rdil dissects of the colonies t right ngles to ech other. Percentge of inhiition ws determined reltive to the growth of the control. Hrvest tuer/stem disese ssessment nd yield: At hrvest ll plnts were crefully removed long with their tuers nd wshed in running wter to remove most of the soil dhering to the surfce. Tuers nd elow ground stems (2-5cm elow soil line) were visully ssessed for oth the incidence nd severity of C. coccodes using the keys shown in ppendix 1. Tuers were lso visully rted s ) infected with C. coccodes surrounding the stolon end, or ) infected with C. coccodes ut not surrounding the stolon end. Tuers from ech pot were counted nd weighed into size ctegories of 3-8g (chts), 8-2g (smll), 2-35g (medium), 35-45g (lrge) nd >45g (oversize). Skin lemishing cused y C. coccodes ws not used s criterion when clculting the mrketle yield. Stem senescence: Plnt senescence ws rted using 5 scle sed on the percentge of folige with wilting, chlorosis, necrosis, or stunting. = %, 1= 1-12%, 2= 13-25%, 3= 26-5%, 4= 51-75% 5= 76-1% Pot experiments: Plnts were grown in 3cm dimeter pots contining 2 litres of 2mm grvel for dringe nd 8 litres of psteurised Mt Compss sndy lom. Plnts were fertilized weekly with solution of N-P-K (Nitrogen ws NH4NO3) prepred t concentrtion of 2 g/l. One seed tuer cv. Colin, with >5% surfce infection y C. coccodes ws plnted in ech pot t depth of 15cm. Experimentl design nd sttisticl nlysis: All trils were set up s Completely rndomised lock designs nd nlysed through GENSTAT V7.. The Lest Significnt Differences (LSD) were clculted y Generl ANOVA unless otherwise specified. 1

17 Pthogen morphology nd ecology Introduction: A numer of studies investigted the morphologicl nd ecologicl vritions mongst isoltes otined from soil, potto plnts nd weed species in Austrli. Some strins my e more ggressive thn others or my e resistnt to certin fungicides. Comining this knowledge with etter understnding of the environmentl conditions ffecting inoculum production y C. coccodes my contriute to the development of improved mngement strtegies for the control of Blck dot on pottoes. Fungl vrition Ojective: To determine the vrition in morphologicl chrcteristics mong isoltes of C. coccodes from different sources in Austrli. Mterils nd Methods: 28 isoltes of C. coccodes from potto tuers (SA), 1 from soil smples (SA, WA, Vic, Ts) nd 2 from weeds (Chenopodium lum Ft hen, Citrullus colocynthis Wild tomto ) were plted onto NP1 selective medi. After 1 dys incution t 24 o C scleroti nd conidi size were mesured nd the presence of sete nd erilly or prostrte myceli recorded. Results nd Discussion: Isoltes produced four distinct vritions in sclerotil formtion (Tle 1). 1. Lrge nd smll lck scleroti, sete, men conidi length of 23.1 nd width 4.5um. 2. Smll olong lck scleroti, sete, men conidi length of 17.5 nd width 3.6um. 3. Lrge, round, lck scleroti, sete, men conidi length of 21.2 nd width 3.7um. 4. Smll round lck scleroti, no sete, men conidi length of 16.4 nd width 3.1um. All conidi were cylindricl with otuse ends, hyline nd septte. However lengths nd widths vried etween isoltes (15.4 um to 25.6um) nd (2.5 um to 5.8um) respectively. All isoltes produced sete except those from WA. Those isoltes tht produced only smll scleroti (vg 125um) generlly cme from SA nd WA, whilst isoltes from Tsmni nd Victori produced oth lrge nd smll scleroti on the sme plte (vg 354um). The ctul role of sete is unknown (12). Similr studies in the UK (41) found no ovious differences in culture morphology etween isoltes of C. coccodes. However significnt difference occurred in sensitivity to the fungicides TBZ nd Fenpiclonil. These results show tht isoltes of C. coccodes re highly vrile in colony morphology, with differences in conidi size nd shpe, presence of sete. Overses studies lso show tht significnt differences occur etween fungicide sensitivity, pthogenicity nd optiml growth temperture (5) Reserch in Isrel hs lso found four different vegettive comptiility groups (VCGs) mongst isoltes collected from the Netherlnds, Frnce nd Isrel (51). Two of these VCG1 nd VCG2 were recovered from ll three countries wheres VCG3 nd VCG4 were not recovered from the smple from Frnce. One reson why this my hve occurred could e tht these VCGs hve dpted to specific regionl climtic conditions. Further studies re required to determine if ny vriility exist mong Austrlin isoltes of C. coccodes such s VCGs, virulence, pthogenicity or fungicide resistnce, so tht the most effective control mesures cn e implemented. 11

18 Tle 1: Morphologicl chrcteristics of C. coccodes collected from SA, Vic, Tsmni nd WA Isolte # Origin Loction Dte isolted Morphologicl chrcteristics on NP1 medi Size of scleroti (um) Size of conidi L X W um Prostrte or eril myceli 45 Potto tuer Vic Lrge & smll lck scleroti, sete X 4.3 P 86 Potto tuer Vic Lrge & smll lck scleroti, sete X 4.4 P 47 Potto tuer Tsmni Lrge & smll lck scleroti, sete X 4.7 P 47 Soil Tsmni Lrge & smll lck scleroti, sete X 4.4 P 65 Soil Tsmni Lrge & smll lck scleroti, sete X 4.5 P 66 Soil Tsmni Lrge & smll lck scleroti, sete X 4.1 P 66 Potto tuer Tsmni Lrge & smll lck scleroti, sete X 3.2 P 12 Potto tuer Tsmni Lrge & smll lck scleroti, sete X 4.6 P 29 Potto tuer Vic Lrge & smll lck scleroti, sete X 3.5 A 77 Soil* SA 7/2/97 Lrge & smll lck scleroti, sete X 4.2 A 2 Potto tuer SA 7/2/97 Lrge & smll lck scleroti, sete X 4.5 A 21 Potto tuer SA Lrge & smll lck scleroti, sete X 4.5 P 2 Potto tuer SA Lrge & smll lck scleroti, sete X 5.8 P 57 Potto tuer SA Lrge & smll lck scleroti, sete X 4.8 P 58 Potto tuer* SA Lrge & smll lck scleroti, sete X 4.6 P 15 Potto tuer SA 6/3/97 Lrge & smll lck scleroti, sete X 4.6 P 16 Potto tuer SA Lrge & smll lck scleroti, sete X 4.7 P 3c Chenopodium lum Ft hen, SA 21/4/1 Lrge & smll lck scleroti, sete X 4.6 P 4c Citrullus colocynthis Wild tomto SA 21/4/1 Lrge & smll lck scleroti, sete X 4.8 P *Isoltes used for inoculum studies. 12

19 Tle 1 cont: Morphologicl chrcteristics of C. coccodes collected from SA, Vic, Tsmni nd WA Isolte # Origin Loction Dte isolted Morphologicl chrcteristics on NP1 medi Size of scleroti (um) Size of conidi L X W um Prostrte or eril myceli 38 Potto tuer Vic Smll olong lck scleroti, sete X 3.2 P 39 Potto tuer Vic Smll olong lck scleroti, sete X 3.4 P 9 Potto tuer SA Smll olong lck scleroti, sete X 2.5 P 9 Soil SA Smll olong lck scleroti, sete X 3.9 P 81 Soil SA Smll olong lck scleroti, sete X 3.1 P 82 Soil SA Smll olong lck scleroti, sete X 3.4 P 6 Soil SA Smll olong lck scleroti, sete X 3.4 P 76 Soil SA Smll olong lck scleroti, sete X 3.8 P 63 Soil SA Smll olong lck scleroti, sete X 4.1 A 11 Potto tuer SA 19/2/97 Smll olong lck scleroti, sete X 4.3 A 12 Potto tuer SA Smll olong lck scleroti, sete X 4.6 P 17 Potto tuer* SA Smll olong lck scleroti, sete X 3.1 P 26 Potto tuer SA Smll olong lck scleroti, sete X 3.3 P 1 Potto tuer SA Lrge, round, lck scleroti, sete X 4.2 P 22 Potto tuer SA Lrge, round, lck scleroti, sete X 4. P 29 Potto tuer Vic Lrge round, lck scleroti, sete X 3.5 A 38 Potto tuer Vic Lrge, round, lck scleroti, sete X 3.2 P 53 Potto tuer WA smll round lck scleroti X 2.5 P 54 Potto tuer WA smll round lck scleroti X 3.2 P 55 Potto tuer WA smll round lck scleroti X 3. P 56 Potto tuer WA smll round lck scleroti X 3.5 P *Isoltes used for inoculum studies. 13

20 Effects of temperture Ojective: To determine the effect of tempertures on the growth of C. coccodes nd disese development. Mterils nd Methods: In-vitro: Preliminry investigtions of morphologicl chrcteristics of C. coccodes on potto plnts reveled some vriility mong isoltes from SA, WA, NSW, Ts nd Vic. Three dissimilr isoltes (77, 58, 17) otined from nturlly infected soil or pottoes (s previously descried in Tle 1) were used. To determine the effects of temperture on the growth of C. coccodes, four replicted 9 cm dimeter petri pltes contining NP1 selective medi, were inoculted with 5mm gr plug tken from the outer edge of 1 dy old culture from ech of the originl three isoltes. Inoculted pltes were grown t 5 tempertures rnging from o C. Rdil growth nd the production of scleroti nd size of scleroti were recorded (s previously descried) t 7-dy intervls over period of 6 weeks. In-vivo: Seed tuers infected with C. coccodes were plnted into pots (s previously descried) with 5 replictes per tretment nd grown in growth rooms t tempertures of 15 o C, 25 o C nd 35 o C with rtificil illumintion (11uEm2sec1) t 16hrs light nd 8 hrs drk. Plnts were wtered y weight every 3 dys to mintin constnt soil moisture content of 6% wter holding cpcity. At 3 weeks fter complete senescence (91 dys fter plnting), plnts nd tuers were hrvested nd visully ssessed for oth incidence nd severity of C. coccodes s previously descried. Results nd Discussion: In-vitro: The rte of growth of C. coccodes ws highest t 24 o C nd lowest t 12 o C (Fig 1). At every time of mesurement, the reltive increse of growth t different tempertures ws similr, except for 32 o C, where fter 21 dys the colony growth slowed nd rdil growth dropped from 2 nd lrgest to 4 th lrgest. Scleroti were lrgest t 16 o C compred to ll other tempertures, whilst the sclerotil density ws highest t 28 o C (Fig 2). Figure 1: Effect of temperture on the rdil hyphl growth of C. coccodes in-vitro oc 8 16 oc 7 24 oc 6 28 oc Figure 5 2: Effect of temperture on the numer nd size of C. coccodes in-vitro. 32 oc rdil growth (mm) Dys fter inocultion Tretments with the sme letter re not significntly different from one nother (LSD P=.1) c d e 14

21 Figure 2: Effect of temperture on the numer nd size of C. coccodes in-vitro scleroti/cm Numer Size c c e d c c scleroti size (um) 12 oc 16 oc 24 oc 28 oc 32 oc Temperture Tretments with the sme letter re not significntly different from one nother (LSD P=.1). In-vivo: C. coccodes did not develop on tuers grown t 15 o C wheres t 25 o C nd 35 o C. It developed on 32 nd 18% of tuers respectively (Fig 3). At 15 o C C. coccodes developed on 39% of stems whilst t 25 o C nd 35 o C ll plnts were infected. Disese incidence nd severity on stems ws lowest t 15 o C (Fig 3). Tuers grown t 25 o C hd the highest level of disese, ut there ws no difference etween mounts of stem infection t the two higher tempertures. Figure 3: Effect of temperture on the incidence nd severity of C. coccodes on tuers nd stems % incidence Tuer Incidence Stem Incidence Tuer Severity Stem Severity ** c 15 oc 25 oc 35 oc Temperture A Severity rting scle to 4:, no diseses; 1, <2%; 2, 3-1%; 3, 11-3%, 4, >3% of tuer surfce ffected. Tretments with the sme letter re not significntly different from one nother (LSD P=.1) Severity Temperture hd significnt ffect on the development of C. coccodes, s the disese ws most severe on tuers nd stems over 25 o C. This explins the field oservtions of incresed disese incidence during summer months when soil tempertures exceed 25 o C t 2cm depth (5yr vg Loxton 15

22 1995-2, Fig 4). Also t higher tempertures there re lrge numers of smll scleroti, creting more colonies nd incresing the inoculum potentil. This study showed tht the optimum temperture (24 o C) for hyphl growth of C. coccodes ws similr to recent findings from Isrel nd the UK (41, 52). The reduced ctivity of C. coccodes t lower tempertures (<15 o C) my correspond with reduction of nutrient utilistion from the host plnt, so the fungus uses the colonized tissue more slowly resulting in ltent infections. The production of scleroti nd susequent infection of plnt tissue occurred over wide rnge of tempertures ut ws gretest t 28 o C nd 32 o C. This reltionship etween sclerotil density nd temperture concurs with recent reserch in Isrel (52). Whilst symptoms were not oserved on tuers t 15 o C high incidence of infected stems were found t this temperture. Whilst temperture influenced the numer of sclerotil produced, other fctors such s ertion nd light re lso likely to exert n effect on sclerotil germintion. Studies hve indicted tht myceliogenic nd sporogenic germintion of scleroti occurs over wide rnge of tempertures (61, 67). However conidil nd mycelil production on scleroti pper to e more sensitive to ertion nd light thn to temperture (52). This suggests tht conidi my e produced more undntly on scleroti on or ove the soil surfce thn on scleroti uried in soil. Scleroti ove the soil surfce would e exposed to higher concentrtion of sunlight nd therefore higher source of conidil inoculum redily ville for dissemintion y rin splsh. In conclusion, disese development ws gretest t tempertures of 25 o C or higher soil temperture, incresing with incresing soil tempertures. By compring this informtion with the soil tempertures in prticulr region, growers my e le to djust their plnting/hrvesting times to reduce the risk of infection, for exmple in the Loxton region of SA, growers should void plnting or hrvesting crops mturing in Jnury nd Ferury (Fig 4). Figure 4: Optimum tempertures for hyphl growth nd sclerotil production for the fungus C coccodes in reltion to the 5 yer verge soil temperture t 1-2cm depths t Loxton m 3.pm Optimum temperture rnge for hyphl growth nd sclerotil production. Overll temperture rnge for the growth of C. coccodes 3 25 Temperture C Jn Fe Mr Apr My Jun Jul Aug Sep Oct Nov Dec 16

23 Disese development Ojective: To monitor the development of C. coccodes on potto tuers nd roots. Mterils nd Methods: Seed tuers cv. Colin with 67% of tuers nturlly infected with C. coccodes were plnted on new ground (cly lom) in the Adelide Hills of South Austrli. Tuers were plnted 24cm prt in rows 8cm prt in n re 1m long nd 28m wide. Every 2 weeks, commencing 7 weeks fter plnting, 4 plnts from 25 eqully spced loctions (totl of 1) were dug up using W smpling pttern nd ssessed for senescence, internl nd externl incidence nd severity of C. coccodes (s previously descried). Results nd Discussion: C. coccodes ws first detected in the vsculr system of stems 42 dys fter plnting, wheres externl symptoms were first detected on stems 7 dys fter plnting. The increse of sp, internl nd externl infections followed similr rte of exponentil development (Fig 5). Tuer infection ws not detected until 84 dys fter plnting, ut lso incresed exponentilly to round 8% of tuers showing externl symptoms of infection t hrvest (Fig 5). The severity of tuer infection followed similr rte of development, however severity of stem infection ws vrile (Fig 6). C. coccodes ws detected in stems of plnts with no visile signs of infection from 56 dys fter plnting (Fig 5). The incidence of internl infection in stems with vile C. coccodes ws vrile, nd my e relted to the efficcy of the surfce steriliztion technique on the externl scleroti, or nturl smpling vrite. While potto seed tuers cn e primry source of inoculum of C. coccodes, the mens y which this inoculum moves from tuer seed to dughter tuers is unknown. This study shows tht C. coccodes first develops on the stem tissue within 6 weeks fter plnting, efore tuer skin infections re ovious round 14 weeks fter plnting. Similr trils hve shown stems, stolons nd roots developing from infected seed were infected with C. coccodes 6 8 weeks fter plnting (4, 41), further suggesting tht this is due to systemic growth of the fungus from infected seed. As with USA studies (37), tuer infections were first oserved t the stolon end suggesting tht conidi produced on stem nd root tissue re crried long the stolon from the mother tuer seed onto progeny tuers. At 84 dys, severity of C. coccodes nd incidence of oth externl nd internl (sp) incresed in conjunction with the incidence of tuer infection t the stolon end. However very few tuers were infected t this dte. It is not until 28 dys fter this initil oservtion tht oth tuer incidence nd severity egin to increse (128 dys). These results suggest tht vritions over time in the incidence of C. coccodes found in the elowground nd ove ground plnt prts my reflect distinct phses of the disese (32, 33) 17

24 Figure 5: Incidence of C. coccodes on plnts grown from nturlly infected tuers. % incidence Stems externl Stems internl (vsculr) Stems internl (sp) Tuers externl Dys fter plnting Figure 6: Severity of C. coccodes on stems nd tuers of plnts grown from nturlly infected tuers. 4 stem Severity 3 2 tuer Dys fter plnting Figure 7: Incidence of tuer stolon infection y C. coccodes. % incidence stolon end infection Dys fter plnting 18

25 Tuer infection from conidi Ojective: To evlute if C. coccodes conidi cn infect mture potto tuers. Mterils nd Methods: Two experiments were conducted to determine if C. coccodes develops on mture tuers inoculted with conidi or microscleroti. In experiment 1, 1 replictes of 1 tuer (cv. Colin) with no evidence of C. coccodes were rtificilly inoculted with 5ml solutions of wter contining either C. coccodes microscleroti (A) or conidi (B). This ws pplied to the tuer surfce using 5ml syringe. Inoculum (A) ws mde y collecting microscleroti from 2 month old cultures (isolte 58) grown on NP1 selective medi. Microscleroti were scrped off the gr surfce nd ground in lender with wter. Inoculum (B) ws prepred y soking 2gms of stems infected with C. coccodes for 3 minutes in 5L wter. Stems were hrvested 5 dys prior to soking, from commercil crop 5 weeks fter the ppliction of Reglone. The concentrtion of oth inoculums (A) nd (B) were djusted to 124cfu/ml using hemocytometer. In experiment 2, 1 replictes of 1 tuer (cv. Colin) with no evidence of C. coccodes were plnted in pots s previously descried. Directly fter plnting tuer, ech pot received 2ml of the solutions descried in Tle 2. The concentrtion of inoculums (B, C, D nd E) were djusted to 124cfu/ml using hemocytometer. 1ml of ech solution ws spred out onto NP1 selective medi to confirm viility. Tle 2: Tretments pplied Tretment A B C D E F Description Wter 23 petri pltes of 2 month old C. coccodes lended with 1L wter 2gms of infected stems soked in 5L wter for 3min As tretment C ut stems spryed with Amistr * 1 week fter Reglone ws pplied. As per tretment C ut the soil surfce of pots ws spryed with Amistr * 5g of stems plced on top of pots nd 5ml wter pplied per dy for 7 dys. * 25g i/h For oth experiments the incidence of C. coccodes colonies on the tuer surfce ws recorded t 7-dy intervls for 28 dys fter the tretments were pplied nd the percentge of infection clculted. Results nd Discussion: C. coccodes ws not oserved on tuers in either experiment until 4 weeks fter inocultion. At this stge colonies 5 1mm dimeter were oserved on tuers inoculted with conidi or microscleroti (Fig 8). C. coccodes developed on ll ten tuers within ech tretment except the uninoculted control. Severity of infection ws less with conidil inocultion compred to microscleroti inocultion. These results show tht tuers cn e infected with conidi of C. coccodes wshed through the soil from the soil surfce. Tuers ecme infected with spores pplied either directly to the soil surfce or wshed from infected stem pieces on the soil surfce. Applying Amistr either to the stem pieces or directly onto the soil surfce did not prevent tuer infection ut significntly reduced the numer of tuers infected nd the severity of tuer infections (Fig 9). This suggests tht folir pplictions of Amistr or other chemicls lte in the seson my e useful technique to reduce inoculum levels from infected hulms. 19

26 Figure 8: Incidence nd severity of C. coccodes on tuers inoculted with microscleroti or conidi. % incidence Microscleroti (A) Conidi (B) Control Tuer Incidence Tuer Severity * A Severity rting scle to 4:, no diseses; 1, <2%; 2, 3-1%; 3, 11-3%, 4, >3% of tuer surfce ffected. Tretments with the sme letter re not significntly different from one nother (LSD P=.1). c Severity Figure 9: Incidence nd severity of C. coccodes on tuers plced in soil where microscleroti or conidi ws pplied to the soil surfce. % incidence Microscleroti (C) Conidi (B) Conidi from Conidi + Amistr stems treted on soil surfce w ith Amistr (D) (E) c Tuer Incidence Tuer Severity * c c Infected stems on top of soil (F) c d Control (A) Severity A Severity rting scle to 4:, no diseses; 1, <2%; 2, 3-1%; 3, 11-3%, 4, >3% of tuer surfce ffected. Tretments with the sme letter re not significntly different from one nother (LSD P=.1). 2

27 Pthogenicity of C. circnnis Ojective: To evlute if C. circnnis ( pthogen of onion) is pthogenic to potto. Mterils nd Methods: Replictes of 1 pots per tretment (s previously descried) were rtificilly infested with microscleroti t concentrtions of 2-microscleroti/g soil of either C. circnnis or C. coccodes. C. circnnis inoculum ws mde y collecting infected onions from commercil grower in the Riverlnd district of South Austrli. One 5mm core of infected skin tissue ws plted on NP1 selective medi (X 2 pltes) nd incuted for 3 dys t 22 o C in the drk. Microscleroti of oth species ws scrped off the gr surfce from 3 dy old cultures, ground in lender with wter, nd the concentrtion djusted using hemocytometer. Microscleroti were thoroughly mixed with ir dried soil t concentrtions of 1 microscleroti/g of soil, then diluted with the sndy lom to chieve the desired concentrtion. One certified mini tuer cv. Colin, with no ovious C. coccodes infection on the tuer surfce, ws plnted in ech pot t depth of 15cm. Pots received 1.3 litres of wter every 3 rd dy, nd were fertilised s previously descried. All plnts were crefully removed from the pots long with ttched tuers nd visully ssessed for incidence nd severity of Colletotrichum sp, nd the incidence of Colletotrichum sp in plnt tissue ssessed s previously descried. Where externl infection ws oserved, plnt mteril ws isolted onto NP1 selective medi nd incuted s previously descried. The identity of cultures ws verified y compring sporultion nd culturl chrcteristics typicl of the species (6, 64) nd with originl isoltes. Results nd Discussion: Although C. coccodes did not develop on tuers grown in the control tretments, it ws isolted from the stems nd sp. It ws lso recovered from some stems, sp nd roots of some tuers inoculted with C. circnnis, indicting tht either the mini tuers were infected efore plnting or tht some other mens of contmintion occurred. Where mini tuers were grown in soil infected with C. coccodes, 6% of the dughter tuers developed lck dot nd the fungus ws isolted from roots, stem nd stem sp. On the other hnd no tuers developed lck dot when grown in soil infested with C. circnnis. C. circnnis ws not recovered from tuers, stem tissue or sp of plnts grown in soil infested with C. circnnis, showing tht the onion pthogen does not infect potto tuers. Tle 3: Incidence of C. coccodes or C. circnnis on inoculted potto plnts. Plnts inoculted with: Not inoculted C. coccodes C. circnnis Plnt prt % infection C. coccodes C. circnnis C. coccodes C. circnnis C. coccodes C. circnnis Roots 1 1 Tuers 6 Stem tissue Stem sp

28 Weeds Ojective: To identify plnts tht host C. coccodes in South Austrli. Mterils nd Methods: Between 21 23, 1 weeds of ech species shown in tle 4 were collected t rndom from pddocks tht hd grown pottoes the previous yer. Plnts were dug up nd then exmined visully for the presence of C. coccodes on stems nd roots. The smpling region included the South Est, Murry Mllee nd Adelide Hills regions of South Austrli. Results nd Discussion: C. coccodes ws found on either the stems or roots of eight species shown in tle 1. Tle 4: Presence of C. coccodes on 28 weed species in SA Fmily Scientific Nme Common Nme Presence of Blck dot Pocee Lolium rigidium Annul ryegrss No Pocee Hordeum leporinum Brley grss No Cucuritcee Citrullus lntus Bstrd melon No Solncee Solnum nigrum Blck nightshde Yes Zygophyllcee Triulus terrestris Cltrop No Astercee Arctothec clendul Cpeweed No Chenopodicee Chenopodium lum Ft hen Yes Astercee Conyz onriensis Flxlef flene No Borgincee Heliotropium europeum Heliotrope Yes Lmicee Mrruium vulgre Horehound No Cryophyllcee Silene petl Mllee ctchfly No Asphodelcee Asphodelus fistulosus Onion Weed No Cucuritcee Cucumis myriocrpus Pddy melon No Ongrcee Oenther glziovin Primrose No Solncee Solnum esurile Quen Yes Brssiccee Diplotxis tenuifoli Snd rocket No Brssiccee Cpsell urs-pstoris Shepherds purse Yes Astercee Chondrill junce Skeleton weed Yes Chenopodicee Slsol kli Soft roly poly No Astercee Dittrichi grveolens Stinkwort No Polygoncee Emex ustrlis Three cornered jck No Brssiccee Sinpsis rvenis Wild mustrd No Pocee Aven ftu Wild ots No Brssiccee Rphnus rphnistrum Wild rdish No Cucuritcee Citrullus colocynthis Wild tomto Yes Brssiccee Brssic tournefortii Wild turnip No Brssiccee Rpistrum rugosum Wild turnip No Polygoncee Polygonum viculre Wire weed Yes This survey hs shown tht C. coccodes survives on the stems nd/or roots of eight common weeds in SA. Severl of the species re lso listed s hosts in the USA (55) eg Solnum nigrum nd Cpsell urs-pstoris. Although the pthogenicity of C. coccodes isolted from weeds ws not tested on pottoes, it is possile tht uncontrolled weed popultions could support nd increse inoculum levels etween rottions or my ct s source of primry inoculum for susequent potto crops. Growers should e le to develop etter disese mngement prctices through selective weed control nd non-host rottionl crops. 22

29 Two of the species identified re from the sme fmily solncee whilst the remining cme from different fmilies. In severl cses, C. coccodes ws not oserved on other species within the sme fmily. This survivl of C. coccodes inoculum in soil is not just restricted to weed hosts ut lso occurs with volunteer pottoes left over from hrvest. These sprout the following yer nd then serve s host for the pthogen nd other diseses. In recent survey of potto pddocks in South Austrli, up to 5, volunteer plnts/h were found one yer fter production (dt not shown) nd in the USA 237,5 tuers/h remined in the field fter hrvest (2). The Austrlin survey lso showed tht volunteer tuers remin vile for up to 4 yers fter the initil crop. The control of these volunteers nd of weed hosts is crucil to reducing the primry inoculum for susequent potto crops. 23

30 Pthogen nd plnt interction Introduction: These studies were undertken to gin etter understnding the pthogen infection cycle nd how it intercts with the host plnt. The im ws to help growers nd reserchers to improve chemicl nd/or culturl methods of control y specificlly trgeting susceptile stges of the disese. Generl mterils nd methods: Internl infection: The stem end of tuers ws sliced nd where stining ws ovious in the vsculr tissue, 2 pieces of pprox 1mm 2 were removed with sclpel nd plted onto NP1 selective medi. After 14 dys incution t 24 o C, pltes were exmined microscopiclly nd scored for the presence or sence of C. coccodes. Mini seed tuers: These were stored in moist vermiculite for pproximtely 3 mo t 22 o C until dormncy ws roken. Tuers were kept under the ove conditions until sprouts.5-1cm in length hd developed. Sprouted tuers were then stored t 5 o C until 2-5 dys efore plnting when they were gin wrmed to 22 o C. 24

31 Tuer infection points Ojective: To determine the incidence of internl (vsculr) nd externl (skin) infections of C. coccodes on tuers. Mterils nd Methods: In 21 nd 22, smple of 1 potto tuers ws collected t rndom from ins of 28 commercil potto properties. Properties were selected t rndom with no prticulr is in respect to seed source or vriety. However, tuers were tested only where the origin of the seed lot could e identified nd ws ccompnied with certificte of registrtion. All tuers were ssessed for oth the incidence nd severity of C. coccodes s previously descried. However only tuers from the 22 smpling were ssessed for internl incidence of C. coccodes. Dt ws nlysed y Spermn s rnk correltion coefficient. Results nd Discussion: Seed lots collected in 21 showed rnge of infection levels. While 61% of the lots were infected, most (28%) hd less thn 5% infection nd none hd more thn 3% (Tle 5). In 22 there ws drmtic increse, with 93% of seed lots infected nd ll hd over 3% of tuers with C. coccodes. No correltion ws found etween the infection on the tuer surfce nd internl infection (r =.18, P =.1) in 22 (Tle 7). However ll tuers with internl infections lso hd externl infections (Tles 7), nd strong correltion exists etween the individul severity rtings nd the % of tuers infected internlly (r =.7, P =.1). Although the importnce of vsculr infection of C. coccodes in seed tuers hs not een determined this spect is of concern s vsculr infections re unlikely to e controlled y treting infected tuers with fungicides pplied to the tuer surfce. Tle 5: Detection of C. coccodes on seed tuers during 21 nd 22. % of tuers in which C. coccodes ws detected. # of seed lots 21 % of seed lots 21 # of seed lots 22 % of seed lots Totl % infected Similr surveys conducted in the USA (37) nd in the UK (58) showed tht the incidence of C. coccodes in certified seed tuers rnged from 9% nd 75% respectively. These studies confirm tht infected tuer seed is the min mens of introducing C. coccodes into potto production res. South Austrli produces 25% of ntionl production mking it the mjor potto producing stte within Austrli. Approximtely 3, tonnes of imported seed produces 334,697 tonnes of pottoes (Austrli Bureu of sttistics 22). Improved understnding of the extent of this disese on seed tuers from different seed producing regions my help in developing more efficient disese mngement strtegies. Results could e used to compre culturl prctices nd environmentl fctors in res tht exhiit different levels of the disese or the plnting of seed tuers from frms with reltively low levels of infection. 25

32 Tle 6: Certified seed survey (21) % tuers infected with C. coccodes externlly nd the verge severity.. Vriety % Externl C. coccodes % Severity C. coccodes % Internl C. coccodes Colin Colin 4.3 Colin Colin Colin 18.8 Colin 9.5 Desiree Desiree 5.4 Desiree Colin Colin Colin Colin 9.6 Atlntic Atlntic 2.3 Not Assessed Desiree 7.5 Colin Atlntic 3.2 Atlntic 4.3 Atlntic 1.6 Atlntic Colin Colin 16.8 Colin 5.3 Colin 2 1. Colin Colin 1.2 Colin 2.2 Avg

33 Tle 7: Certified seed survey (22) % tuers infected with C. coccodes either externlly/internlly nd the verge severity.. Vriety % Externl C. coccodes % Severity C. coccodes Colin Colin Colin Desiree Colin 1 3. Bison Kenneec Colin Desiree Colin Colin Colin Colin Desiree Desiree Atlntic Colin Atlntic Ruy Lue Ruy Lue 1 2. Colin Colin Colin. Colin 1. Colin Colin Desiree 1 2. Colin Avg % Internl C. coccodes 27

34 Light on C. coccodes seed Ojective: To evlute the effects of light on the viility of C. coccodes on seed tuers. Mterils nd Methods: 4 seed tuers with more thn 75% of their surfce re infected with C. coccodes were removed from cold storge fter 2 months t 4.5 o C. Tuers were then plced in the drk t room temperture rnging from (16 24 o C) for 3 dys. After this time two 5mm cores were removed from the stolon end of ech potto nd plted onto NP1 selective medi. 2 of these tuers were then exposed to nturl sunlight t mient tempertures (16 24 o C) whilst the remining 2 tuers were kept in the drk in similr tempertures. After 19 dys, two 5mm cores contining C. coccodes microscleroti were removed from ech potto nd plted onto NP1 selective medi. All 4 tuers were then plnted into pots, wtered nd fertilized s previously descried. All pltes were incuted for 14 dys t 22 o C in the drk, fter which the sclerotil viility ws determined s previously descried. After 3 months growth, ll dughter tuers nd stems from ech pot were ssessed for incidence of C. coccodes s previously descried. Results nd Discussions: The exposure of potto seed tuers to light induces the formtion of green pigmenttion on the surfce of the potto. In this experiment ll tuers tht were exposed to light ecme green, indicting n increse in the presence of glycolkloids. Ninety percent of scleroti were vile on tuers removed from cold storge. The viility ws reduced to 75% nd 54% when tuers were kept for period of 19 dys in the drk or light respectively. These differences hd little influence on disese development on dughter tuers s oth the incidence of infected stems nd tuers were similr on potto plnts grown from tuers sujected to either the light or drk tretments. Whilst the exposure of seed tuers to light showed no effect on the viility of C. coccodes it my e eneficil to seed growers. Plnting seed tht hs een exposed to light hrdens sprouts nd dvnces the physiologicl stge giving n erlier emergence, nd erlier mturity. This is dvntgeous where lrger numer of smller tuers re desirle. In ddition this erly mturity reduces the time where crop is susceptile to virus infections. Since greening of tuers is strongly ffected y the qulity, durtion, nd the light intensity s well s temperture, further trils need to conducted to fully evlute the effects of greening on tuer orne disese Figure 1: Effect of light on the viility of C. coccodes recovered from tuers exposed to light. % viility Just out of storge 19 dys in drk 19 dys in light c vile scleroti stems infected tuer infected Tretments with the sme letter re not significntly different from one nother (LSD P=.1). 28

35 Systemic infection Ojective: To estlish if seed orne C. coccodes is systemic. Mterils nd methods: 3 tuers, cv Colin, with more thn 3% of their surfce ffected y C. coccodes were plnted into pots s previously descried (1 st August 23), 1 tuer per pot. Eighteen dys fter plnting (19 th August 23) 2 pots were selected t rndom nd the plnts crefully dug up. Hlf the stems from ech plnt were cut t 2.5cm from the mother seed tuer, nd the other hlf cut t 5cm. Stems were then replnted into new pots contining sterile soil nd the mother seeds discrded. All pots were wtered nd fertilized s previously descried. Two months fter plnting stems nd dughter tuers of ll tretments were ssessed for the externl presence of C. coccodes. In ddition, plnts tht were not seprted from the mother seed 18 dys fter plnting, hd internl isoltions from the min stolon t 1, 2, 3, 4 nd 5cm from the mother seed tuer nd t 1, 2, 3, 4, nd 5cm from soil line (green tissue) nd sp extrcted from stem tissue t 1, 2, 3, 4 nd 5cm from the soil line. Results nd Discussion: The incidence of C. coccodes on tuers ws significntly reduced when the min root system ws cut either 2.5 or 5. cm ove the mother seed compred to uncut root systems (6%, 4% nd 1% respectively). Disese incidence in stems followed similr pttern, with 2%, 5% nd 74% respectively (Fig 11). Stem nd stolon infection reduced with incresing distnce from the infection source, ut ws still detectle. The incidence of C. coccodes in stolon tissue significntly decresed from 4% t 1 nd 2cm to 2% t 3 nd 4cm nd % t 5cm wy from the mother seed. C. coccodes ws recovered from the sp of ll stems collected from 1 to 5 cm ove the soil line. No C. coccodes ws isolted from internl stem tissue in ny of the tretments. In other experiments C. coccodes ws recorded from tissue efore sp, nd there ws no pprent reson for the nil detected in tissue in this experiment. It is unlikely tht externl growth of C. coccodes exceeded 2.5 or 5. cm within 18 dys fter plnting. It is more likely tht the infections of C. coccodes developed from tissue colonized internlly. These results show tht infection from the mother seed exerted continul influence on the infection of progeny, nd tht infection from the mother seed strts soon fter plnting. The erly infection of plnt tissue suggests tht this is due to systemic growth of the fungus from the infected seed piece. Figure 11: Incidence of C. coccodes on tuers nd stems grown from infected tuers or plnt tissue tht ws isolted from stem tissue either 2.5cm or 5.cm ove the mother tuer. % incidence Mother seed Tuer Incidence 2.5cm wy from mother seed Stem Incidence Tretments with the sme letter re not significntly different from one nother (LSD P=.5). 5.cm wy from mother seed 29

36 Figure 12: Incidence of C. coccodes in stem sp nd stolon tissue t 1 5cm wy from either the mother seed or soil line (cv. Colin). % incidence Internl stolon tissue Stem sp 1 1cm from soil line/mother seed 2cm from soil line/mother seed 3cm from soil line/mother seed 4cm from soil line/mother seed c 5cm from soil line/mother seed Tretments with the sme letter re not significntly different from one nother (LSD P=.5). 3

37 Systemic crry over nd disese trnsfer through soil. Ojective: To evlute the development of C. coccodes on dughter tuers grown from tuers infected either externlly, internlly or oth nd to determine if tuers cn e infected if plnted djcent to infected tuers. Mterils nd Methods: Two experiments were conducted using certified seed tuers, cv. Colin. Tuers were ssessed for the incidence of C. coccodes oth externlly nd internlly s previously descried. They were then plced into the ctegories of externl, internl, externl + internl or no visile externl infection. All tuers in the externl incidence ctegory hd greter thn 5% of their surfce ffected y C. coccodes. In the first experiment, 1 tuers within ech ctegory descried ove were plnted into pots on the 17 th Novemer. In the second experiment, 1 tuers within ech ctegory descried ove (except those with internl nd externl infection) were plnted on the sme dy into trys (45cm x 3cm x 25cm), plced 25cm prt from tuers with no visile sign of C. coccodes. All plnts were wtered nd fertilized s previously descried. After 3 months growth, ll dughter tuers nd one stem selected t rndom from ech pot/try ws visully ssessed for oth incidence nd severity of C. coccodes s previously descried. Tuers ssessed in the second experiment were those produced from the clen mother seed. Results nd Discussion: The incidence of dughter tuers with externl infection ws lowest where mother seed hd no visile signs of C. coccodes. However the level of infection ws ove 6%, suggesting tht the mother seed ws infected with C. coccodes ut not detected visully (Fig 13). The incidence of stem infection y C. coccodes ws significntly lower where mother seed hd either no visile sign of C. coccodes or only internl C. coccodes (oth 5%) compred to those with either externl or externl plus internl incidence of C. coccodes (61% & 82% respectively). Oservtions of greter stem infections developing in plnts grown from seed tuers in which C. coccodes ws detected compred to those grown from seed tuers with no C. coccodes hs previously een oserved.(37). Seed tuers with externl infection produced dughter tuers with the highest incidence nd severity of infection, s well s stem infection nd numer of tuers ffected t the stolon end. This indictes tht while internl infections cuse disese in dughter tuers, externl skin infections re of more significnce, nd hve greter effect on the totl disese levels t hrvest. Figure 13: Effect of externl or internl infection of C. coccodes on the development of disese in dughter tuers. Shdehouse tril, Lenswood 22 (Expt 1) % incidence Affected t stolon end Stems Incidence No visile infection Externl infection Internl infection Externl & internl infection Seed tuer infection 31 Tuer Incidence Tuer Severity Severity

38 The incidence of C. coccodes on dughter tuers, percentge ffected t stolon end nd numer of stems ffected ws significntly lower when seed tuers were plnted longside seed with no visile sign of C. coccodes compred to those plnted longside infected seed (Fig 14). Similr levels of disese developed on tuers nd stems of plnts grown ner seed tuers with internl or externl infection. However the severity of tuer infection ws less with internlly infected tuers. These results show tht propgules of C. coccodes move through the soil from infected seed tuers to infect dughter tuers of djcent plnts. Figure 14: Development of C. coccodes on tuers nd stems of plnts plnted 25cm wy from infected tuers Shdehouse tril, Lenswood 22. (Expt 2) % incidence Tuer Incidence Affected t stolon end Stems Incidence Tuer Severity No visile infection Externl infection Internl infection Severity 32

39 Potto cultivrs Ojective: To evlute cultivr susceptiility to C. coccodes. Mterils nd Methods: Fourteen commercil potto cultivrs were grown on commercil property in the Murry Mllee region of SA (8 fresh mrket, 3 crisp nd 3 french fry vrieties). The tril ws plnted on 14/2/1, plots were doule rows y 5 m long, replicted 5X, rows 86cm prt nd set spcing 28cm. 2 months fter complete senescence tuers from ll tretments were hnd dug from rndomly chosen 2m sections in oth rows. These were plced into cold storge for 28 dys fter which su smples of 1 tuers from ech replicte were ssessed for oth incidence nd severity of C. coccodes s previously descried. Results nd Discussion: All cultivrs were susceptile to C. coccodes, with Colin, the most widely used cultivr for wshed pottoes, one of the most susceptile (Tle 8). Overses studies hve shown tht thin-skinned cultivrs, such s mny chipping cultivrs, re more susceptile to C. coccodes thn thicker-skinned ones (3). These results indicte tht the tuer infection ws lower in the crisping nd frying vrieties, ut stem infection ws not similrly ligned. Significnt differences were oserved in the incidence of stem coloniztion etween cultivrs nd the severity of tuer surfce infection (Tle 8). Differences etween stem nd tuer infection ws oserved in some cultivrs, eg Desiree hd the lowest level of stem infection, ut one of the highest levels of tuer infection. Reserch from the UK hs shown tht there re significnt differences etween cultivrs for resistnce to C. coccodes. Severity of infection ws greter in the erly cultivrs such s Mris Brd compred to the min crop cultivrs such s Romno nd Shelgh. This ws ttriuted to the fct tht the erly cultivrs set tuers efore those of the min crop cultivrs which mens tht tuers re in contct with soil inoculum for longer period. In ddition, vrition did occur within oth erly nd lte cultivrs, suggesting tht there is genetic influence. Further trils on Austrlin vrieties re required to estlish these differences nd ssist in producing cultivrs with improved disese resistnce. If resistnce gene is identified then it my e possile to incorporte this into those cultivrs suitle for the fresh mrket. 33

40 Tle 8: Incidence of C. coccodes coloniztion on fourteen cultivrs of potto grown in the Murry Mllee district of SA, 22 Vriety % of infected stems % of infected tuers Disese severity Skin type Use Desiree 2 e 76 e 3.7 thin Fresh Sonic * 5 cd thick Crisp Ruy Lou 5 cd 68 d 3.6 thin Fresh Winter Gem 6 c thin Fresh Ndine 6 c 79 e 3.1 thin Fresh Dwmor 7 55 c 2.5 thick Crisp Shine 7 59 c 2.6 thick Fresh Fontenot 8 69 d 3.2 thin Fresh Atlntic thick Crisp Riverin Russet netted Fry Id Rose 8 59 c 2.8 thin Fresh Russet Burnk(CS) netted Fry Shepody netted Fry Colin 1 76 e 3.3 thin Fresh A Severity rting scle to 4:, no diseses; 1, <2%; 2, 3-1%; 3, 11-3%, 4, >3% of tuer surfce ffected. Tretments with the sme letter re not significntly different from one nother. 34

41 Inoculum levels Ojective: To evlute the effect of different soil inoculum levels on the development of C. coccodes on potto tuers nd roots Mterils nd Methods: Replictes of 3 pots per tretment were rtificilly infested with C. coccodes microscleroti t concentrtions of, 5, 1 nd 2 microscleroti/g soil. Inoculum ws mde y collecting microscleroti from 1 month old cultures (isolte 58) grown on NP1 selective medi. Microscleroti were scrped off the gr surfce, ground in lender with wter, nd the concentrtion djusted using hemocytometer. Microscleroti were thoroughly mixed with ir dried soil t concentrtions of 1 microscleroti/g of soil, then diluted with the sndy lom to chieve the desired concentrtions. One certified mini tuer cv. Colin, with no ovious C. coccodes infection on the tuer surfce, ws plnted in ech pot t depth of 15cm. Pots received 1.3 litres of wter every 3 rd dy nd were fertilised s previously descried. 5 plnts selected t rndom from ech tretment were visully ssessed for senescence every 2 weeks strting 7 weeks fter plnting. All plnts tht were ssessed for senescence were crefully removed from the pots long with ttched tuers nd visully ssessed for oth the incidence nd severity of C. coccodes on tuers nd stems nd the incidence of C. coccodes in plnt tissue (s previously descried). Results nd Discussion: Senescence developed t similr rtes in ll tretments until 56 dys fter plnting where it incresed in ll inoculted tretments (Fig 15). Plnts grown in 2-microscleroti/g soil senesced more rpidly thn the plnts grown in the lower levels of inoculum. At 98 dys fter plnting, C. coccodes hd developed on 47% - 64% of tuers nd 1% of stems on plnts grown in inoculted soil (Fig 15). Whilst overses studies (1,15) showed C. coccodes severity on tuers incresed s inoculum incresed, there were no significnt differences on the levels of C. coccodes infection on tuers grown in soil with different levels of inoculum. However oth the internl stem incidence nd severity incresed with incresing inoculum levels. Plnts grown in soil with 5 nd 1cfu/g soil hd significntly lower levels of C. coccodes in sp ( nd 2cfu s respectively) compred to those grown in 2cfu/g (458 cfu s/ml) (dt not shown). Figure 15: Rte of senescence in Colin potto plnts grown in soil with different inoculum levels. % senescence Control 5 cfu 1 cfu 2 cfu Dys fter plnting Figure 16: Effect of soil inoculum level on the incidence nd severity of C. coccodes on tuers nd stems (externl nd internl) t 96 dys. 35

42 % incidence c c Tuer incidence Tuer severity Stem externl Stem internl incidence incidence Cfu s per gm soil Stem severity Tretments with the sme letter re not significntly different from one nother (LSD P=.5) Severity In this study the rnge of inoculum densities of C. coccodes used ws similr to those found in soils where pottoes hve een grown in the Mllee region of South Austrli. Whilst study in the USA (4) hs shown tht infected tuers re highly correlted with colony forming units (cfu s) found in the soil, no differences in incidence or severity of tuers or externl stem infection were detected in the plnts grown t different inoculum levels in this experiment. Internl vsculr infection of stems nd disese severity on stems were the only fctors to vry with different levels of inoculum. Previous studies using popultions rnging from ( 1 microscleroti/gm dry soil) found significnt differences in infection levels etween the different densities (9, 1). Further studies need to e done s 1 microscleroti/g soil my e ove the threshold for cusing significnt levels of disese. Therefore inocultion with more of the lower concentrtions my show the threshold where disese will occur. 36

43 Wter nd hulm disese development. Ojective: To evlute the effects of overhed irrigtion fter hulm senescence on the incidence of C. coccodes. Mterils nd Methods: Certified seed tuers cv. Colin nturlly infected with C. coccodes were plnted t two sites on 12 th Decemer 23 in the Mllee region of SA. At site one, tuers hd 48% incidence nd 1.45 severity nd t site two 56% incidence nd 1.56 severity. Reglone ws pplied (3.5L/H) to site one (15 th Mrch) nd site two (7 th April). Two dys lter plstic tents were plced over single rows covering 6 plnts (Fig 17). These were plced in rndomised lock design nd replicted 7 times. Plstic ws supported t lest 3cm ove plnts to llow ir movement nd to reduce ny greenhouse like effects. In ddition the sides of the tents were uried t lest 3cm elow the soil line so tht ny run off wter ws diverted wy from the tuers. Overhed irrigtion vi centre pivot occurred on verge twice week with pprox 4mm pplied t ech wtering. Six weeks fter the ppliction of Reglone the middle two plnts from ech tent nd their tuers were hrvested nd then ssessed for incidence nd severity of C. coccodes (s previously descried). Figure 17: 3cm Results nd Discussion: In the Mllee re of SA, potto plnts re often irrigted fter hulm desicction to reduce skin lemishes cused from excessive hot, dry soils nd to ssist with the hrvesting process. In this tril reducing exposure to the irrigtion significntly reduced incidence nd severity of C. coccodes developing on tuers (Figs 18, 19). At oth sites, C. coccodes developed on more thn 7% of tuers wtered fter desicction wheres round 3% were infected in the covered tretment. These results further confirm the role of overhed wtering dispersing conidi from infected tissue through the soil onto dughter tuers. The ppliction of wter t this stge lso increses the potentil infection levels s free wter or reltive humidity of 1% (6) is needed for conidil germintion. The high incidence of stolon end infection compred to ud end infection in this experiment hs lso een oserved in severl other experiments in this report nd y overses reserches (37). Conidi re stimulted to dhere to the plnt tissue (tuers/roots) in response to host lechtes. Since they do not function s survivl structures their viility declines rpidly if wter is withheld or high tempertures occur (6, 45). Once the conidi penetrte the host tissue, the fungus will survive unseen etween the wx lyer nd the cuticle structures (ltent infection). Figure 18: Incidence of C. coccodes on dughter tuers (Site 1) Tuer Incidence 9 Affected t stolon end

44 Figure 19: Incidence of C. coccodes on dughter tuers (Site2). % incidence Tuer Incidence Affected t stolon end Tuer Severity Severity 1 Wter pplied fter dessiction Wter withheld fter dessiction Tretments with the sme letter re not significntly different from one nother (LSD P=.5). 38

45 Reglone/Dye Ojective: To evlute if wter pplied to desiccted plnt stems ove ground cn trnsfer spores on to the min root system. Mterils nd Methods: A commercil crop of pottoes cv. Colin grown in the Murry Mllee ws spryed with the desiccnt Reglone (15/12/3). Eight dys lter, different rtes of wter contining 5% solution of florescent dye ws pplied to individul plnts nd to the se of plnts using wtering cn (Tle9), with 5 replictes of ech tretment (1 plnt = 1 replicte). Tle 9:Rtes of wter nd zone of ppliction. Tretment Appliction zone Dye rtes Irrigtion rtes A Plnt folige 5L/H - B Plnt folige 5L/H 5ml per m2 C Plnt folige 2L/H - D Plnt folige 2L/H 5ml per m2 E Soil t plnt se 5L/H - F Soil t plnt se 5L/H 5ml per m2 G Soil t plnt se 2L/H - H Soil t plnt se 2L/H 5ml per m2 Results nd Discussion: Fluorescent dye ws detected on the roots of potto plnts in ll tretments. Where wter volumes of 2L/h were pplied, most of the roots were contminted with fluorescent dye. In the tretment where the lowest rte 5L/h of wter ws pplied only 4% of the roots were detected with fluorescence. These results show tht high volumes of wter pplied to the soil penetrtes the soil long the edge of the stem nd moves long the stem to the roots rther thn perculte through the soil to the roots. The usul mount of wter pplied y centre pivot irrigtion is pprox 4 5mm (4-5ml/m2). This would wet the soil to pprox cm in depth, whilst second irrigtion cycle would wet the soil to pprox 1cm in depth. These volumes of wter re pplied frequently to crops tht hve mtured during the summer months of South Austrli. These results suggest tht high volume of wter pplied to potto crops post desicction or ner hrvest is likely to wsh conidi of C. coccodes from infected stems nd move them long the stem nd roots to infect stolons nd dughter tuers prior to hrvest. 39

46 Irrigtion Ojective: To evlute the effects of overhed nd susurfce irrigtion on the incidence of C. coccodes on dughter tuers. Mterils nd Methods: Seed tuers, cv. Colin, with more thn 5% of their surfce ffected y C. coccodes were selected from certified seed. One tuer ws plnted in ech pot t depth of 15cm on the 1 st August 23. Replictes of 5 pots were plced into growth room t temperture of 25 oc with rtificil illumintion (11uEm2sec1) t 14hrs light nd 8 hrs drk photoperiod. All pots received 1.3 litres of wter every 3 rd dy nd were fertilized s previously descried. 5 pots received overhed irrigtion vi wtering cn nd 5 pots received susurfce irrigtion y plcing the wter in try t the se of ech pot. After 3 months, ll dughter tuers from ech pot were ssessed for incidence nd severity of C. coccodes s previously descried. Results nd Discussion: The incidence nd severity of stolon end infection were significntly less on plnts wtered from the se of pots compred to overhed wtering (Fig 2). More thn 9% of tuers irrigted with overhed wtering developed lck dot compred to 55% where plnts were wtered susurfce. Similrly the severity of the tuer infection nd the numer of tuers ffected t their stolon end ws significntly higher in tuers grown from plnts wtered overhed compred to those wtered susurfce. Since inoculum ws only present on the originl seed piece these results suggest tht wter moving downwrds through the soil plys significnt role in moving inoculum from the infected tuer seed to the dughter tuers. Figure 2: Effect of overhed or susurfce wtering on the incidence nd severity of C. coccodes on dughter tuers. % incidence Affected t stolon end Tuer Incidence Tuer Severity Severity Overhed wtering Susurfce wtering Tretments with the sme letter re not significntly different from one nother (LSD P=.5). 4

47 Pivot vritions Ojective: To mesure the incidence nd severity of C. coccodes on tuers collected long the rdius of centre pivot irrigtion system. Mterils nd Methods: Certified seed tuers cv. Colin nturlly infected with C. coccodes (48% incidence & 1.45 severity) were plnted t 4ton/H on 12 th Decemer 23 in the Mllee region of SA. Reglone ws pplied (3.5L/H) on 15 th Mrch. The tril site consisted of 12 plots (35m X 2 rows) running from the centre point to the outer point. Six weeks fter the ppliction of Reglone, 2 plnts nd their tuers were hrvested from ech plot using W smpling pttern cross 2 rows within ech plot. These were then visully ssessed for oth incidence nd severity of C. coccodes (s previously descried). Results nd Discussion: These results show tht oth the incidence nd severity of C. coccodes infection ws highest t the centre of the pivot where most wter is pplied nd lowest t the extremity of the pivot where the lest wter is pplied to the crop (Fig 21). A similr study on the incidence of Lte Blight Tuer Rot showed similr pttern of infection in tht the highest level of disese developed close to the pivot centre where most wter ws pplied (35). Figure 21: Incidence nd severity of C. coccodes on tuers from plnts grown t vrious distnces from the pivot centre. 1 Tuer Incidence Tuer Severity 4 9 % incidence Severity Distnce from centre of pivot (m) 41

48 MANAGEMENT Introduction: Surveys of certified seed plnted in SA showed high incidence of C. coccodes. This infected seed is the primry source of introducing the disese into new potto growing res. There is need to develop effective tretments to minimise this primry spred of the disese from seed to progeny tuers. Generl mterils nd methods: Source of test mteril: Cold pressed deftted mustrd seed mel or pellets contining 25% mel of the cultivr Brssic junce were otined from commercil outlets nd used s the source of glucosinoltes. Soil fumigtion: Fumignts were pplied using Rumsted, 5 lded injection unit (Pictures 4 nd 5 Pge 73-74) set t depth of 25cm. Methm Sodium (32.7% i of sodium methyldithiocrmte) ws pplied t 7L/h nd Telone II 2L/h (114g i of 1,3-dichloropropene). Within 4 hours fter chemicl ppliction, 5mm of wter ws pplied to the tril res. Orgnic mendments: Amendments of seed mel, pellets nd freeze dried plnt smples were plced in 2cm dimeter vessels nd hydrted with wter on v/w sis (5ul/.1g). Controls were exposed to 2ul of wter lone. These were plced in inverted petri dishes, which ech contined 5mm disc of mycelium tken from the mrgin of 5 7 dy old fungl colony of C. coccodes (58). The petri dishes were seled with Gldwrp to contin ny voltiles produced (Fig 22). Figure 22: NP1 selective medi 5mm disc of C. coccodes orgnic mendment Seed tretment methods. Seed tuers, cv. Colin, with more thn 5% of their surfce covered y C. coccodes scleroti were selected from commercil grower. Btches of ten tuers were either spryed with fungicide using commercil Controlled Droplet Applictor or dipped in chemicl solution (Tle 1). Irrigtion: Pots were wtered with n utomtic irrigtion system tht pplied 4 litres of wter every 3 rd dy. 42

49 Smpling nd detection of C. coccodes in soil: Soil smples were collected from tril sites using W shped smpling pttern with minimum of 25 eqully spced ssessment sites per field. The size of ech smple re vried due to the different size of tril sites. Soil ws smpled with 2.5cm dimeter corer used to remove t lest 1g-soil smple. Smples were ulked nd mixed y rotting the soil in the plstic g in which smples were collected. A 5g-soil smple ws ir dried t room temperture (15 to 25 o C) for 2 weeks to eliminte short-lived propgules such s conidi nd mycelil frgments. After drying, the soil ws homogenised nd sieved through 85, 5 nd 25 micron sieves. The resulting soil smple ws nlysed for the presence of C. coccodes y either of two methods: Method 1: Plting out five su smples of.1mg onto seprte pltes of NP1 selective medi, using n Anderson Smpler (9). Method 2: 4 gm of dried soil ws dded to 4ml of.2% wter gr nd 1ml of the solution spred over the surfce of 5 pltes of NP1 selective medi. Pltes from oth methods were incuted t 24 o C in the drk for 7 14 dys nd then exmined under ifocl microscope for colonies typicl of C. coccodes. The numer of colony forming units (CFU) g of unseived soil ws clculted using the formul (X/.1) x Y/2, where. Totl weight of unseived soil Totl weight of soil plted out Totl weight of sieved soil Totl numer of Cfu s on plte = 5gms =.1gms = Y = X Assessments: Tuers from ech pot or the centre 2m of oth rows in ech the plot were hnd dug, wshed, counted nd weighed into size ctegories of 3-8g (chts), 8-2g (smll), 2-35g (medium), 35-45g (lrge) nd >45g (oversize). Skin lemishing cused y C. coccodes ws not used s criterion when clculting the mrketle yield. Disese levels were ssessed s previously descried. Tle 1: Fungicides evluted s tuer seed tretments Tretment Active ingredient Concentrtion (%) Rte of product/1kg tuers Seed spryed* Amistr 5g/kg zoxystroin gm Filn 5g/Kg Bosclid gm Crio 25g/kg Pyrclorostroin gm Dithne 75g/kg mncoze gm Fungflor 75g/kg imzil.75 2 gm Mxim 1g/L fludioxonil ml Octve 462g/Kg prochlorz gm Tecto 5g/L thiendzole ml Seed dipped** Formlin 4g/L formldehyde 4. Sporekill 12g/L didecyl dimethyl NH4CI 1. Chlordox 2% chlorine dioxide 85% phosphoric cid.5 nd.1 * Applied in 2L wter ** Dipped for 15 minutes nd rinsed with wter once Untreted tuers were dipped in wter for 15 min. 43

50 Biofumigtion Inhiition from voltiles relesed from Brssic sp Ojective: To evlute the ntifungl properties of voltiles relesed from three different Brssic species, mustrd mel, mustrd pellets nd one ot species ginst C. coccodes. Mterils nd Methods: Smpling green mnure crops: Seed of B. junce, B. npus, Rphnus stivus nd Ot cv Drummond were purchsed from commercil supplier nd plnted in commercil potto field t Woodside, South Austrli in Autumn. At 1% flowering one hundred plnts were dug up t rndom from ech cruciferous plot to depth of 15cm. Ot plnts were collected from five 1m 2 res when kernels were t milky stge (Feekes scle 11.1). Soil ws wshed from the roots of ll plnts nd su smple of 5 plnts from ech smple seprted into lef, stem nd root tissue nd immeditely freeze-dried. Dried mteril ws weighed, ground nd stored in seled vils t 2 o C. Anlysis of glucosinoltes: A 3mg smple of freeze-dried Brssic mteril otined from root, stems or leves. Levels of glucosinoltes (GLS s) were nlysed using grdient HPLC method y Dr J Kirkegrd (CSIRO Plnt Industry Cnerr). Mycelil growth in the presence of voltiles relesed from orgnic mendments: Seed mel, pellets (s previously descried) nd freeze dried plnt smples rnging from to.1gm were hydrted nd plced in petri dishes, contining C. coccodes isolte 58 (s previously descried) nd replicted 7X. Pltes were incuted t 22 o C in the drk for up to 11 dys nd the percentge inhiition of fungi clculted s previously descried. Results nd Discussion: Glucosinoltes (GSL's) relesed from Brssic residues re hydrolized y endogenous myrosinse (3) to produce rekdown products, one of which is the voltile compound isothiocynte (ITC). These re highly iocidl to diverse rnge of orgnisms including nemtodes, cteri, fungi, insects nd germinting seeds (8,38,54,72). With ll isoltes of C. coccodes, voltiles from lef nd root tissue were more suppressive thn those from stem tissue nd lef tissue lone, B. junce nd B. npus were more inhiiting thn R. stivus (Fig 23). Figure 23: Inhiition of mycelil growth fter exposure to voltiles relesed from Brssic residues t different rtes nd concentrtions of ITC lierting GLS. % inhiition B junce "leves" B junce "roots" B junce "mel" B junce "pellets" B npus "leves" B npus "roots" R stivus "leves" R stivus "roots" ots Dose rte (gm) 44

51 GLS levels were highest in the mel, where 48 umol/g were relesed (Tle 11). The next highest levels of 38 umol/g were relesed from the leves of B. junce nd the lowest 2.1 umol/g from R. stivus stem tissue. GLS levels were highest in lef tissue compred to root or stem tissue from B. junce, however levels were higher in root in ll other species. The lowest levels in ll plnts were found in stems with levels rnging from 2 to 6 umol/g. Tle 11: Concentrtions of ITC lierting GLS. B. junce Mel B. junce Pellets Orgnic mendment B. junce lef, (root) B. npus lef, (root) R. stivus lef, (root) Ots lef GLS (umol/g) (16) 25 (36) 6 (12) This work shows tht voltiles emnting from B. junce mel or B. junce nd B. npus leves nd roots re inhiitory to C. coccodes. All Brssic plnt mterils tested suppressed fungl growth, ut the response vried etween plnt prts. For exmple B. junce mel completely inhiited growth of C. coccodes wheres lef tissue only inhiited fungi etween 23 52%. Suppression generlly incresed with incresing levels of mendment. The higher level of suppression cused y the B. junce mel extrcts compred to the pelletised formultions re consistent with the higher ITC concentrtions found in the mel. This study nd others (59) show tht glucosinoltes vry mong Brssic species nd even cultivrs within the sme species. They lso vry etween plnt prts, growth stge nd lso chnge with environment. In ddition Ros (59) lso showed tht time of exposure needed to control pthogen growth differs etween fresh nd dried mendments. Fresh cge required n extr 1 dys to chieve the sme degree of control of F. oxysporum f. sp. conglutinns compred with dried cge. 45

52 Hydrtion effects on voltiles relesed from B. junce Ojective: To evlute the effects of vrying hydrtion periods on the nti fungl properties of voltiles relesed from deftted mustrd seed mel ginst C. coccodes. Mterils nd Methods: Amendments of.1gm mustrd seed mel were hydrted for, 2, 12, 24 or 48hrs efore eing plced in petri dishes contining C. coccodes isoltes (77, 58, 17), s previously descried. Ech plte ws replicted 6 times. Pltes were incuted t 22 o C in the drk for up to 11 dys nd the percentge inhiition of fungi ws clculted. To determine if the tretments were fungitoxic, mendments were removed fter 298 hrs nd 5 dys lter were inspected to see if hyphl growth hd incresed. Results nd Discussion: Voltiles relesed from B. junce seed mel were inhiitory to the growth of C. coccodes (Fig 24), ut not lethlly, s hyphl growth recommenced once the mel ws removed. The effect of the seed mel on hyphl growth decresed over time, nd with incresing prehydrtion time. A possile reson for this is tht once certin isothiocyntes ecome hydrolized they dissipte more redily into the tmosphere, reducing the concentrtion of ny llelopthic compounds. Figure 24: Inhiition of C. coccodes fter exposure to voltiles relesed from B junce seed mel. % inhiition Unhydrted mel hrs prehydrtoin 2hrs prehydrtion 12hrs prehydrtion 24hrs prehydrtion 48hrs prehydrtion Wter Exposure (Hours) 46

53 Efficcy of voltiles relesed from B. junce seed mel in soil Ojective: To evlute the efficcy of voltiles relesed from B. junce seed mel on C. coccodes t vrying depths in potting soil. Mterils nd Methods: Mt Compss sndy lom mixed with or without mustrd seed mel t the equivlent rtes of 1, 2 nd 3 ton/h ws plced into PVC cylinders (1cm dimeter y 38cm height), 6 cylinders per mel concentrtion. Six individul muslin gs ech one contining 5gm of inoculted vermiculite were plced t 7, 14 nd 21cm depths, in ech tue. Inoculnt ws prepred y grounding 1 month old C. coccodes cultures (isolte 77) on NP1 selective medi with wter nd the concentrtion djusted to 2 cfu/ml using hemocytometer. 2 ml of the resulting solution ws then dded to 6 gms of vermiculite nd hnd mixed to provide uniform distriution of the inoculum. Cylinders were then plced upright onto dringe rcks nd 8mls of wter dded to hydrte the mel. The tops of the cylinders remined open. After 192 hrs the muslin gs were removed nd 2 pieces of Vermiculite from ech g plted onto NP1 selective medi. Inhiition of the individul fungi ws determined y the percentge of vermiculite pieces tht grew C. coccodes. Results nd Discussion: Both 2 nd 3 ton/h of seed mel hd significntly higher levels of inhiition t 14 nd 21cm thn ll other tretments or depths (Tle 12). Inhiition incresed with incresing depth, nd with incresing concentrtion of mel. One possile reson inhiition incresed s the depth incresed is tht GLS (minly 2-Propenyl nd 3- Butenyl) in the upper profile dissipted into the tmosphere more redily whilst GLS in lower levels were confined for longer periods due to the incresed soil mtter ove cting s sel. The vilility of isothiocyntes in soil hs een shown to depend on the nture of the soil (59). Thus, soils with higher levels of cly or orgnic mtter re likely to enefit less from Brssic mendment thn lighter soils. Tle 12 Effect of lechtes from Mustrd Mel, on the viility of C. coccodes uried t different depths fter 192hrs exposure. % Inhiition Control 1 ton 2 ton 3 ton 7cm cm cm c 15.1 LSD 2.2 LSD 1.9 LSD 2.1 Men (LSD = 3.5) c Tretments with the sme letter re not significntly different from one nother (LSD P=.5). 47

54 Chemicl control Invitro ssessment of tuer seed tretments Ojective: To evlute in vitro vrious fungicides pplied s potto tuer seed tretments for the control of C. coccodes. Mterils nd Methods: Two lortory experiments were conducted to evlute the efficcy of vrious fungicides s tuer seed tretments. Seed tuers were treted with fungicides t rtes s previously descried. Tuers were ir dried for 15 2 minutes nd then plced on plstic trys. After incution for 7 dys in the drk t room temperture, five 5mm cores contining C. coccodes microscleroti were removed from ech potto nd plted onto NP1 selective medi. After 14 dys incution t 22 o C in the drk the percentge inhiition of rdil growth nd microscleroti density were determined (s previously descried). In the second experiment, the more effective chemicls were re-exmined t 3 different rtes (Tle 13) on different seed source ut with similr incidence of C. coccodes. Tuers were lso incuted for 7 dys in the light. 48

55 Tle 13: Chemicls nd rtes pplied Tretment Active ingredient Concentrtion (%) Rte of product/h/ 1kg tuers Seed spryed* Amistr 5g/kg zoxystroin.48 2.gm.4 1.6gm.2.8gm Crio 25g/kg Pyrclorostroin.4 3.4gm.3 2.4gm.2 1.6gm Dithne 75g/kg mncoze.22 6.gm gm.11 3.gm Fungflor 75g/kg imzil 1.5 4gm gm.75 2gm Mxim 1g/L fludioxonil ml ml ml Octve 462g/Kg prochlorz.14 6.gm gm.7 3.gm Seed dipped** Formlin 4g/L formldehyde Chlordox 2% chlorine dioxide 85% phosphoric cid 2. 2ppm 1. 1ppm.1 1ppm * Applied in 2L wter ** Dipped for 15 minutes nd rinsed with wter once Untreted tuers were dipped in wter for 15 min. Results nd Discussion: In experiment one, tuer seed tretments of Mxim, Octve nd Crio completely inhiited mycelil growth of C. coccodes (Fig 25). Growth ws inhiited to vrious degrees y other tretments ut C. coccodes ws not inhiited with Tecto treted tuers. Amistr ws effective t much lower doses thn ll other chemicls in reducing hyphl growth. Mxim nd Octve were the most effective t inhiiting microscleroti whilst inhiition of microscleroti ws 5% or less in ll other tretments (Fig 26). 49

56 Figure 25: Inhiition of mycelil growth of C. coccodes from cores of skin tken from tuers treted with fungicides (Expt 1). % inhiition d d 2 1 de e e Mxim Crio Octve Dithne Fungflor Amistr Formlin Sporekill Chlordox 5ppm Chlordox 1ppm Filn Tecto Untreted Tretments with the sme letter re not significntly different from one nother (LSD P=.5). Figure 26: Inhiition of C. coccodes microscleroti density produced from cores of skin tken from tuers treted with fungicides on selective medi, (Expt 1). % inhiition of cfu's Mxim c d e e e e e e e Octve Chlordox 5ppm Chlordox 1ppm Formlin Crio Tecto Fungflor Amistr Sporekill Dithne Filn Tretments with the sme letter re not significntly different from one nother (LSD P=.5). The overll inhiition ws less in experiment 2 (Figs 27, 28) thn experiment one, possily due to different isolte of C. coccodes (eg. different seed source) or tht incution period took plce in high humidity trys under light. 5

57 Figure 27: Inhiition of mycelil growth of C. coccodes from cores of skin tken from tuers treted with fungicides, (Expt 2). % inhiition Fungflor 1.5 Fungflor Fungflor 1.5 Fungflor 1.25 Fungflor.75 Crio.4 Crio.3 Crio.2 Mxim 1.25 Mxim.93 Mxim.625 Dithne.22 Dithne.17 Dithne.11 ChlorDox/Oxine 2ppm ChlorDox/Oxine 1ppm ChlorDox/Oxine 1ppm 51 Octve.14 Octve.1 Octve.7 Formlin 5 Formlin 4 Amistr.48 Amistr.4 Amistr.2 Figure 28: Inhiition of C. coccodes microscleroti density produced from cores of skin tken from tuers treted with fungicides on selective medi, (Expt 2). % inhiition Fungflor.75 Crio.4 Crio.3 Crio.2 Mxim 1.25 Mxim.93 Mxim.625 Dithne.22 Dithne.17 Dithne.11 ChlorDox/Oxine 2ppm ChlorDox/Oxine 1ppm ChlorDox/Oxine 1ppm Octve.14 Octve.1 Octve.7 Formlin 5 Formlin 4 Amistr.48 Amistr.4 Amistr.2 As dose rtes of Fungflor, Mxim nd Dithne incresed, mycelil growth decresed. All remining tretments did not show strong dose response. Only Fungflor showed dose response to microscleroti production. As in experiment 1, Mxim inhiited microscleroti production completely t ll rtes whilst inhiition incresed when dose rtes decresed with oth Chlordox nd Amistr. This my hve een due to the fungicides inititing chnge in the fungus so tht it ecme more tolernt.

58 Effect of fungicides on trnsmission in storge Ojective: To determine if chemicl tretments pplied to the mother seed influenced the levels of tuer infection y C. coccodes on the dughter tuers during cold storge. Mterils nd Methods: Dughter tuers from the previous experiment, grown from treted seed, were ssessed for the incidence nd severity of C. coccodes (s previously descried) prior to eing plced in storge t 4.5 o C. After period of five months the level of C. coccodes on tuers ws ressessed. Results nd Discussion: The incidence or severity of C. coccodes did not chnge significntly within ny of the tretments fter 5 months storge t 4.5C (Figs 29, 3). However significnt decrese in the incidence nd severity occurred within the control tuers. This ws possily due to n increse in the incidence of Silver Scurf, which cn msk regions ffected y C. coccodes when visully inspected. In ll tretments Silver scurf incidence incresed y 79% or more; eg: untreted tuers incresed from 21% to 1% (dt not presented). Figure 29: Effect of chemicl tretments on the incidence (% tuers ffected) of C. coccodes on the cultivr Colin t hrvest nd 5months fter cold storge, Lenswood % incidence Before storge After storge 2 1 SporeKill Amistr WG Octve Mxim Crio Control Figure 3: Effect of chemicl tretments on the severity of C. coccodes on Colin t hrvest nd 5 months fter cold storge, Lenswood Before storge After storge severity 2 1 SporeKill Amistr WG Octve 52 Mxim Crio Control

59 Effect of fungicides on crry over of seed. Ojective: To demonstrte the introduction of C. coccodes into the soil on tuer seed nd to determine the survivl in soil. Mterils nd Methods: In August 22, seed tuers cv. Colin, with more thn 5% of their surfce ffected y C. coccodes were treted with fungicides t rtes s previously descried. Tuers were left to ir dry for 15 2 minutes efore plnting. Infected tuers were plnted one per pot nd 15cm deep into pots with ech tretment replicted 1X. Pots were wtered nd plnts fertilized s descried in generl mterils nd methods. Plnts were grown for 2 months, fter this time, plnts nd tuers from ech pot were hrvested nd ssessed for oth incidence nd severity of C. coccodes s previously descried. The soil ws left fllow in the pots for 12 months with no wter or fertilizer tretments. In Novemer 23, certified mini tuers of the vriety Kenneec were plnted into the sme pots. After 3 months growth, dughter tuers were ssessed for oth incidence nd severity of C. coccodes s previously descried. Results nd Discussion: In most tretments, the incidence nd severity of infection ws highest in the second crop grown from untreted mini tuers (Figs 31, 32), even where good control ws chieved with the initil tretments of Mxim nd Octve. These results show tht infected seed tuers, whether treted or not re primry source of introducing inoculum into the soil, where it estlishes itself nd eventully serves s soil-orne inoculum for future crops (31, 37, 56). Crio significntly reduced the levels of C. coccodes in the following yer, with only 5% infection. Only Amistr completely inhiited infection developing in the 2 nd plnting. Results lso confer with UK studies in tht scleroti cn survive for periods of one yer or more in soil (22). More work needs to e undertken to determine the most pproprite tretments to reduce soil inoculum to cceptle levels nd hence rottion scheduling. Figure 31: Effect of chemicl tretments on the incidence (% tuers ffected) of C. coccodes, on the cv. Colin Shdehouse tril, Lenswood 22/3 % incidence Dithne DF Tecto ChlorDox/Oxine 1ppm 1st Plnting 22 2 nd Plnting 23 Filn Fungflor SporeKill ChlorDox/Oxine 5ppm Formlin Amistr WG Figure 32: Effect of chemicl tretments on the severity of C. coccodes, on the cv. Colin Shdehouse tril, Lenswood 22/3 Octve Mxim Crio Control 53

60 1st Plnting 22 2nd Plnting Severity 2 1 Dithne DF ChlorDox/Oxine 1ppm Tecto Filn SporeKill Fungflor ChlorDox/Oxine 5ppm Formlin Amistr WG Octve Mxim Crio Control 54

61 Greenhouse screening of fungicides Ojective: To evlute the effect of fungicides pplied s tuer seed tretments for the control of C. coccodes on stems/tuers. Mterils nd Methods: Experiments were conducted in the greenhouse to evlute vrious fungicides s tuer seed tretments. Seed tuers were treted with vrious fungicides s previously descried. Tuers were left to ir dry for 15 2 minutes efore plnting. One seed tuer infected with C. coccodes ws plnted t 15cm depth into pots, replicted 1X. Pots were wtered nd plnts fertilized s descried in generl mterils nd methods. Insecticides were pplied s needed to control whitefly nd thrips. Plnts were grown for 2 months in greenhouse where the men mximum temperture ws 23.2C nd minimum 15.4C. After this time, plnts nd tuers from ech pot were hrvested nd visully ssessed for oth incidence nd severity of C. coccodes s previously descried. Results nd Discussion: 77% of tuers from plnts grown from untreted tuers were infected with C. coccodes (Fig 33). Amistr, Octve, Mxim nd Crio were the only tretments tht reduced oth the incidence (14, 9, 8, & % respectively) nd severity (.3,.2,.3 &. respectively) of C. coccodes (Figs 33, 34). All other tretments hd levels of infection similr to the untreted control. Amistr, Mxim nd Crio were the only tretments with significntly less stem infection compred to the untreted control (Figs 35, 36). Figure 33: Effect of chemicl seed tretments on the incidence of C. coccodes on dughter tuers cv. Colin. % incidence Dithne DF c c Tecto ChlorDox/Oxine 1ppm c Filn c Fungflor c c SporeKill ChlorDox/Oxine 5ppm c Formlin Amistr WG Octve Mxim Crio Control Tretments with the sme letter re not significntly different from one nother (LSD P=.1). c Figure 34: Effect of chemicl seed tretments on the severity of C. coccodes on dughter tuers cv. Colin c c

62 Tretments with the sme letter re not significntly different from one nother (LSD P=.1). Figure 35: Effect of chemicl seed tretments on the incidence of C. coccodes on plnt stems cv. Colin. c c c c c 1 c c c 9 8 c c % incidence Dithne DF Tecto Filn Fungflor SporeKill ChlorDox/Oxine 1ppm ChlorDox/Oxine 5ppm Formlin Octve Crio Amistr WG Mxim Control Tretments with the sme letter re not significntly different from one nother (LSD P=.1). Figure 36: Effect of chemicl seed tretments on the severity of C. coccodes on plnt stems cv. Colin. 4 3 c c c c c c 56 c c c

63 Tretments with the sme letter re not significntly different from one nother (LSD P=.1). 57

64 Field nd greenhouse evlution of seed nd soil tretments. Tril 1. Ojective: To evlute in Greenhouse nd in vivo, fungicides nd iologicl products for the control of C. coccodes on stems/tuers. Mterils nd Methods: Seed tuers were treted with fungicides s previously descried, t rtes shown in Tle 14. Tuers were left to ir dry for 15 2 minutes nd then five 5mm cores contining C. coccodes microscleroti were removed from ech potto nd plted onto NP1 selective medi nd incuted t 22 o C in the drk for 4 weeks. Percentge inhiition of rdil growth nd sclerotil density were determined fter 28 dys (s previously descried). One seed tuer ws plnted into pot on 17/11/2 (replicted 1X) nd plnts wtered nd fertilized s previously descried. Plnts were grown for 3 months in glsshouse where the men mximum temperture ws 26.4 o C nd minimum 16.7 o C. At 2 weeks fter complete senescence; plnts nd tuers were hrvested from ech pot nd visully ssessed for oth incidence nd severity of C. coccodes (s previously descried). Tle 14: Chemicls/iologicls nd rtes pplied. Tretment Active ingredient Concentrtion (%) Rte of product/1kg tuers Seed spryed* Amistr 5g/kg zoxystroin gm Crio 25g/kg Pyrclorostroin gm Fungflor 75g/kg imzil.75 2 gm Mxim 1g/L fludioxonil ml Octve 462g/Kg prochlorz gm Voom 25 (2 % 2-propenyl-ITC) 5. 5ml in 1.5L wter Voom 25 (2 % 2-propenyl-ITC) ml in 1.75L wter Seed dipped** Formlin 4g/L formldehyde 4. Soil treted Voom 25 (2 % 2-propenyl-ITC) 1. 1L in 19L wter/h Trichogrow 1.5kg/h s drench t plnting (5L wter) nd then every 28 dys Mustrd Mel 1 ton/h1gm/m2 (7.1gm/pot Mustrd Mel 2 ton/h2gm/m2 (14.2gm/pot * Applied in 2L wter ** Dipped for 15 minutes nd rinsed with wter once Untreted tuers were dipped in wter for 15 min. Results nd Discussion: Mxim nd Octve were the most inhiitory to the production of C. coccodes scleroti (Fig 37), however inhiition of mycelil growth ws under hlf tht of sclerotil density. Mxim, Amistr, Crio, Octve nd the 5% rte of Voom provided the est control of tuer incidence nd severity of C. coccodes. (Fig 38) s well s inhiiting the development of C. coccodes t the stolon ends of tuers nd on stems (Fig 39). 58

65 Figure 37: In-vitro inhiition of C. coccodes 28 dys fter treting tuers with different chemicls. Shdehouse, Lenswood, % inhiition Mycelium Sclerotium 2 1 Fungflor Mxim Crio Voom 5% Voom 2.5% Formlin Voom 1% Amistr WG Octve Figure 38: Effect of seed tretments on the incidence nd severity of C. coccodes on dughter tuers (cv. Colin), Lenswood % incidence Tuer incidence Tuer severity Crio Voom 1% Fungflor Trichoderm Formlin Voom 2.5% Mustrd mel 2ton/h Mustrd mel 1ton/h Octve Mxim Amistr WG Voom 5% Control 59

66 Tril 2. Ojective: To evlute in-furrow ppliction of Amistr nd seed tuer tretments of Amistr, Mxim nd Octve for the control of C. coccodes in pottoes. Mterils nd methods: Two trils were conducted on consecutive yers t the Lenswood Centre situted pproximtely 5km est of Adelide on ground tht hd previously een plnted to pottoes nd ws known to e infected with C. coccodes. At site one the level of C. coccodes in the soil ws 36 CFU (colony forming units)/gm of dry soil, ut levels were not mesured preplnting t site two. At oth sites tuers cv. Colin nturlly infected with C. coccodes were used. No tuer disese nlysis ws conducted t site one whilst t site two 43% of tuers were infected with C. coccodes. 1kg tches of tuers were spryed with fungicides (shown in tle 15) using hnd spryer whilst eing rotted in hndoperted cement mixer. Tuers were left to ir dry 1 dy prior to plnting. Amistr ws pplied using motorised ck-ck spryers to the soil in-furrow t plnting just prior (Site one) nd just following (site two) plcement of the seed tuer. The rtes of ppliction re outlined in Tle 14. Site one ws plnted on 11th Decemer 22, whilst site two ws plnted on the 24 th Novemer 23. Both trils were plnted in plots 2 rows wide with 5 replictes of ech tretment. Plots were 6m long (23 seed pieces t 26cm spcing) t site one nd 8m long (32 seed pieces t 25cm spcing) t site two. The treted rows were seprted y 2 nd 1 uffer rows of tuers cv. Colin (site one nd site two respectively), with 2 rrier rows on either edge of the treted rows. The rows were nked t site one on 17 th Jnury 23 nd t site two on the 12 th Jnury 24 nd irrigted s required. The trils were inspected regulrly for folir diseses, ut levels of infection were low nd did not wrrnt sprying. At site one the tril ws llowed to senesce nturlly nd ws hrvested on 23 rd April. Site two ws spryed off with Reglone on the 5 th Mrch nd hrvested on 16th Mrch. Assessment of tuers ws s previously descried. Tle 15: Chemicls nd rtes pplied Tretment Active ingredient Concentrtion (%) Seed spryed* Rte of product/h Rte of product/1kg tuers Mxim 1g/L fludioxonil ml Octve (site one only) 462g/Kg prochlorz gm Amistr (site two only) 5g/kg zoxystroin gm Soil treted Amistr SC ** (site one only) 25g/kg zoxystroin L Amistr SC ** (site two only) 25g/kg zoxystroin L * Applied in 2L wter ** Applied t 4L wter/h Emergence: The numer of plnts emerged per plot were counted t 4 nd 36 dys fter plnting t site one nd site two respectively. 6

67 Hrvest disese ssessments nd yield: Tuers were hrvested nd the yield clculted t oth sites (s previously descried) except tht site two where 6m ws dug. They were then visully ssessed for oth incidence nd severity of C. coccodes (s previously descried). Results nd Discussion: Emergence: Between 73 nd 8% of plnts emerged t site one nd etween 9 to 96% of plnts t site two. At ech site there ws no significnt difference in emergence levels etween tretments. Disese ssessments: At site one, C. coccodes developed on 72% of tuers in the control plots. Whilst Amistr pplied in furrow (drench) significntly lowered levels of C. coccodes tuer infection compred to the control, the Mxim nd Octve seed tretments hd no effect (Fig 39). At site two C. coccodes developed on more thn 96% of tuers in the control. Mxim tretment hd significntly higher levels of tuer infection compred to Amistr pplied s either seed or in furrow tretments (Fig 4). The lowest level of C. coccodes (43%) ws found in the 25g.i./H rte of Amistr pplied s seed tretment. The severity of C. coccodes on the tuers followed similr trend s the incidence, in tht the severity ws highest (>2) in the controls nd lowest (.7) in the 25g.i./H rte of Amistr pplied in furrow. The tril showed tht Amistr provides good control of C. coccodes whether pplied to the tuers efore plnting or to soil in furrow t plnting. In oth of these trils Amistr ws more effective thn Mxim when pplied s tuer seed tretment. Figure 39: Effect of in furrow (drench) fungicide tretment nd seed tretment on the incidence nd severity of C. coccodes on dughter tuers (Site 1). % incidence Tuer Incidence Tuer Severity Severity 1 Mxim seed Octve seed Amistr in-furrow Control Tretments with the sme letter re not significntly different from one nother (LSD P=.1). 61

68 Figure 4: Effect of in furrow (drench) fungicide tretment nd seed tretment on the incidence nd severity of C. coccodes on dughter tuers (Site 2). 1 9 Tuer Incidence 4 % incidence Tuer Severity Severity 1 Mxim seed Amistr seed Amistr in-furrow Control Tretments with the sme letter re not significntly different from one nother (LSD P=.1). Yield: At site one yields were not significntly different etween tretments compred to the control (37.4 ton/h). However yields were highest where Amistr ws pplied in furrow (39.5 ton/h) nd lowest in the Octve nd Mxim tretments (35.6 & 33.9 ton/h respectively). At site two ll yields were less thn the control (25.6 ton/h), Mxim ws the only tretment tht reduced yields significntly (14.3 ton/h). Amistr in furrow nd on seed were 21. nd 17 ton/h respectively. 62

69 Tril 3. Ojective: To evlute tuer seed tretments nd infurrow soil pplictions for the control of C. coccodes on nturlly infected pottoes. Mterils nd Methods: This tril ws plnted on commercil property in Mllee region of SA t 3.5ton/H. Seed tuers were treted with fungicides s previously descried (Tle 1). Tuers were ir dried for 1-2 dys efore plnting. Plots were 1 m long y 2 rows wide with 6 replictes of ech tretment. Treted rows were seprted y 2 uffer rows. The grower mintined the site s per commercil prctice. Two dditionl tretments were included, where fungicides were pplied to the soil in-furrow (drench) t plnting in nd (< 15cm) just prior to plcement of seed tuer (Tle 16). Motorised ck-ck spryers were used to pply oth products. At 3 weeks fter complete senescence tuers were hrvested, the yield clculted nd tuers visully ssessed for oth the incidence nd severity of C. coccodes (s previously descried). Tle 16: Appliction method, rte nd ctive ingredient of fungicide evluted. Tretment Active ingredient Concentrtion (%) Rte of product/h/1kg tuers Soil spryed*** Amistr 5g/kg zoxystroin.62 5 gm Mxim 1g/L fludioxonil L *** Applied in 4L/h Results nd Discussion: Nerly ll of tuers grown from untreted seed were infected with C. coccodes t hrvest (Fig 41). Significntly less infected tuers developed in most tretments where the mother seed ws treted with fungicides. The most effective of these tretments were Mxim, Octve, Amistr nd Crio where 4% or less of tuers were infected with C. coccodes. Figure 41: Effect of fungicide seed tuer tretments on the incidence of C. coccodes on dughter tuers t hrvest. % incidence c c c c c d d de e Filn Tecto Chlordox 1ppm Mxim drench Chlordox 5ppm Fungflor Formlin Sporekill Dithne Amistr drench Amistr Crio Mxim Octve Untreted Tretments with the sme letter re not significntly different from one nother (LSD P=.1). 63

70 Figure 42: Effect of fungicide seed tuer tretments on the severity of C. coccodes on dughter tuers t hrvest. 4 severity c c c c c d d de e Filn Tecto Chlordox 1ppm Mxim drench Chlordox 5ppm Fungflor Formlin Sporekill Dithne Amistr drench Amistr Crio Mxim Octve Untreted Tretments with the sme letter re not significntly different from one nother (LSD P=.1). In-furrow (drench) tretments of Mxim nd Amistr were less effective thn seed tretment in reducing disese incidence or severity (Fig 41, 42). However one overses study hs reported tht the use of zoxystroin s n in-furrow drench were effective (Severity reduced y 6 8% nd incidence reduced y 5 1%) (5). One possile reson my e tht the mjority of dughter tuer infections were from seed orne rther thn soil inoculum. Also the ppliction rtes used in our trils my hve een too low. For exmple in the UK Amistr is pplied t higher rtes gm H (personl comm. S.Wle 24) compred to the rte 25 gm H used in our trils. 64

71 Tril 4. Ojective: To evlute pplictions of Amistr, Mxim, Crio nd Octve for the control of C. coccodes. Mterils nd methods: Certified seed tuers cv. Colin nturlly infected with C. coccodes (1% incidence nd 3 severity) were used in this tril. One tonne tches of tuers were pssed through grder resulting in pprox 3% of tuers eing cut to size. Tuers were treted with fungicides t rtes s outlined in Tle 17. In ddition tuers were coted fter fungicides were pplied in mixture of 5% cement nd 5% fur rk. Tuers were ir dried for 2 dys efore plnting. 2 tuers from ech tretment were collected t rndom nd two C. coccodes microscleroti were removed from ech potto nd plted onto NP1 selective medi. Pltes were incuted t 22 o C in the drk for 2 weeks efore the viility of scleroti ws determined. 1 tuers from ech tretment were evluted in greenhouse tril. One seed tuer ws plnted into pot on 24th Mrch 23, replicted 1 times (s previously descried). Pots were wtered nd plnts fertilized s previously descried. Plnts were grown for 3 months in greenhouse. Remining tuers were plnted on 26 th Mrch 23 in the Mllee region of SA t 4 ton/h. 25 smples of soil rndomly collected from cross the tril site efore plnting indicted level of C. coccodes in the soil of 2 CFU (colony forming units)/gm of dry soil. The tril ws plnted in plots 4 rows wide nd 1m long in rndomised lock design, with 5 replictes of ech tretment. The numer of plnts emerged per plot were counted t 4 dys fter plnting. On the 7 th July, 4 weeks fter complete senescence, plnts nd tuers were hrvested from ech pot nd plot nd yields clculted s descried in generl mterils nd methods in this section. They were then visully ssessed for oth incidence nd severity of C. coccodes (s previously descried). Tle 17: Chemicls nd rtes pplied Tretment Active ingredient Concentrtion (%) Rte of product/1kg tuers Seed spryed* Mxim 1g/L fludioxonil ml Amistr 5g/kg zoxystroin gm Octve 462g/Kg prochlorz gm Crio 25g/kg Pyrclorostroin gm * Applied in 2L wter Results nd Discussion: Assessments 4 dys fter plnting showed tht Mxim pplied to tuer seed significntly reduced tuer emergence compred to the untreted tuers in oth the field nd greenhouse trils y 14% & 13% respectively. Emergence in ll other tretments ws round 9% nd showed no significnt differences compred to the control or etween ech other. Mxim ws the only tretment tht significntly inhiited the germintion of scleroti fter tretment compred to the untreted tuers (% & 5% respectively). All other tretments were not significntly different to the untreted tuers. 65

72 All tretments significntly reduced the incidence nd severity of tuer infection compred to the control in greenhouse trils (Fig 43), ut only Crio significntly reduced the incidence of disese in the field tril (Fig 44). This is possily due to the level of soil infection lredy present in the field. Amistr nd Mxim were the only tretments tht significntly incresed the yield (Fig 45). Figure: 43: Effect of Amistr, Mxim, Crio nd Octve seed tretment on the incidence nd severity of C. coccodes on dughter tuers grown in the green house. 5 Tuer Incidence 4 4 Tuer Severity 3 % incidence Severity Amistr Octve Mxim Crio Control Tretments with the sme letter re not significntly different from one nother (LSD P=.5). Tle 44: Effect of Amistr, Mxim, Crio nd Octve tuer seed tretments on the incidence nd severity of C. coccodes on dughter tuers grown in the field. 5 Tuer Incidence 4 4 Tuer Severity 3 % incidence Severity Amistr Octve Mxim Crio Control Tretments with the sme letter re not significntly different from one nother (LSD P=.1). Yield. 66

73 Figure 45: Effect of Amistr, Mxim, Crio nd Octve seed tretment on the yield of dughter tuers. Yield ton/h Field Greenhouse 1 Amistr Octve Mxim Crio Control Tretments with the sme letter re not significntly different from one nother (LSD P=.1). 67

74 Evlution of fumigtion Tril 1. Ojective: To evlute the effects of vrious fungicides pplied s either tuer seed tretments or in furrow soil tretments, for the control of C. coccodes in soil fumigted with nd without Methm Sodium. Mterils nd Methods: Trils were conducted on two commercil grower properties in the Mllee region pproximtely 25km northest of Adelide. At oth sites soil ws fumigted with Methm Sodium (s previously descried), site one on 7th Novemer nd site two on 8 th Novemer 22 (Pic 1,2,3,4). Seed tuers, cv. Colin were used t oth sites. At site one, 25% of the tuers were infected with C. coccodes nd n verge severity rting of 1.1 wheres site two ll of the tuers were infected with C. coccodes nd n verge severity rting of 2.7. At oth sites chemicls were pplied to whole tuers (s previously descried) t rtes outlined in Tle 18, nd llowed to dry efore storing overnight. At site one, cut seed weighing pprox 42 94gms/ech were treted in tches of 11kg the dy efore plnting (22 nd Novemer) whilst t site two, whole seed weighing pprox 7 12gms/ech were treted in tches of 25kg two dys prior to plnting (11 th Decemer). Filn, Tecto nd Chlordox t.5% were not included; Dithne ws included t higher rte nd dditionl tretments included (Tle 18). In-furrow sprys were pplied t plnting just prior to plcement of the seed tuer using motorised spryers. Both sites hd previously een plnted to pottoes pprox 5yrs go, ut site one ws plnted to cnol lst yer nd site two ws under psture. Soil smples rndomly collected from the tril sites efore plnting indicted tht the levels of C. coccodes t site one were 4.6 cfu s (colony forming units)/gm of dry soil nd 29.6 cfu s/gm dry soil t site two. Site one ws spryed off on the 12 th Mrch nd hrvested on 14 th April, whilst site two ws spryed off on the 18 th Mrch nd hrvested on 23 rd April. Plots t oth trils were 4 rows wide nd 1m long with set spcing of 17.5cm nd row spcing of 85cm t site one nd 8cm site two. Design ws s previously descried, with 4 replictes of ech tretment. The treted rows were seprted y 2 uffer rows of tuers. The tril site ws mintined s per the commercil prctices of the grower. Tle 18: Rtes of fungicides. Tretment Active ingredient Concentrtion (%) Rte of product/h/1kg tuers Seed spryed* Crio 25g/kg Pyrclorostroin gm Dithne 75g/kg mncoze gm Mxim + Sporekill only t site two ml + 2 ml Soil spryed*** Amistr 5g/kg zoxystroin.4 25 gm Mxim 1g/L fludioxonil ml * Applied in 2L wter ** Dipped for 15 minutes nd rinsed with wter once *** Applied in 4L/h Untreted tuers were dipped in wter for 15 min. All in furrow tretments re pplied using Solo motorised ckpck spryers in volume of 4L/H. 68

75 At 3 weeks fter complete senescence, tuers were hrvested nd the yields clculted (s previously descried). Tuers nd stems were then ssessed for oth incidence nd severity of C. coccodes s previously descried. Results nd Discussion: Disese ssessments: At site 1, C. coccodes developed on 44% of tuers in the unfumigted plots compred to 29% in the fumigted (Tle 19). Mxim nd Octve provided the est control in the unfumigted plots, reducing the incidence of disese to 18 nd 16% respectively. The Amistr in-furrow (drench) tretment significntly incresed oth the incidence nd severity of disese compred to control in the unfumigted res, with the highest levels of disese found in this tretment (68% incidence nd 1. severity). In the fumigted res, Fungflor ws the only tretment to mrkedly reduce disese incidence, lthough due to the vriility the differences etween tretments were not significnt. In tril 2, where high inoculum levels were present, the results were extremely vrile, nd disese levels were generlly lower thn in site one with lower soil inoculum. Low levels of disese were found in the control, with 14% nd 1% of tuers infected in the unfumigted nd fumigted plots respectively. Mxim gin provided good control with 9% of tuers infected in the unfumigted tretments. The snitizers (Sporekill, Chlordox /oxine nd Formlin ) lso hd lower levels of disese compred to some other tretments, possily due to controlling the high level of seed infection. Amistr in-furrow (drench) hd high levels of disese, 4% incidence compred to 14% in the control nd 9% with Mxim. In the fumigted re, mny tretments reduced disese levels however Mxim ws not effective, with the highest levels of disese (26%) oserved. Overll, in soil with high levels of inoculum, fumigtion reduced these levels nd the seed tretments provided little dditionl enefit. Where low levels of soil inoculum occur, the seed tretments were more effective in unfumigted soil. Picture 1: Incorportion of green mnure crop (Brssic), four weeks prior to fumigtion, (Site 1). Picture 2: Fumigtion, (Site 1). 69

76 Picture 3: Incorportion of psture, four weeks prior to fumigtion, (Site 2). Picture 4: Fumigtion, (Site 2). Tle 19: Effect of chemicl tretments on the incidence (% tuers ffected) nd severity of C. coccodes, on tuers cv. Colin, grown t sites with high nd low levels of soil inoculum. Tretment low inoculum (Site 1) high inoculum (Site 2) 7