Phomopsis castanea infection in chestnuts from Canterbury, New Zealand

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1 from Cantrbury, Nw Zaland A. Osmonalival, D.L. McNil\ A. Stwartl, D.J. Klinac 2 and K.D.R. Wadia 3 1 Soil, Plant and Ecological Scincs Division, PO Box 84, Lincoln Univrsity, Cantrbury, Nw Zaland 2 Th Horticultur and Food Rsarch Institut of Nw Zaland Ltd., Ruakura Rsarch Cntr, Privat Bag 3123, Hamilton 3 Nw Zaland Chstnut Council Inc., PO Box 1925, Hamilton Abstract This papr dscribs a study to idntify gnotyp and circumstanc diffrncs in th rat, lvls and location of Phomopsis castana (Sacc.) infction during th vgtativ, flowring and arly fruiting stags of chstnuts. Ths diffrncs hav bn studid in both pollinator and main production typ cultivars. This papr rports rsults from th orchard at Lincoln Univrsity during th sason. Th purposs of this rsarch wr to charactris lvls of in diffrnt tr lins, to partially dscrib th pidmiology of th disas and to idntify slction paramtrs to achiv this goal. Ths data suggst that th Cantrbury climat is poorly suitd to Phomopsis as a nut disas though it is wll suitd to Phomopsis as an ndophyt during wintr, arly spring and possibly lat autumn. Additional ky words: pidmiology, suscptibility, slction paramtrs, nut disass, ndophyt Introduction Th chstnut is a larg, dibl sd producd insid a prickly cas, calld a burr. Unlik othr nuts it is high in carbohydrats and moistur and low in oil contnt, so that optimal storag conditions for th nuts ar mor lik that of fruits than othr nuts (Nw Zaland Chstnut Council Inc., 1997) and storag rots play a larg part in dtrmining chstnut quality. Most chstnuts grown in Nw Zaland ar of hybrid origin (McNil, 1997). Howvr, Oraguzi t al. (1998) rportd that South Island slctions wr mostly C. sativa-lik whil North Island slctions wr mor C. crnata-lik. Worldwid, thr ar svr limitations posd to th commrcial cultivation of chstnuts by both th chstnut blight (causd by Endothia parasitica) and th gall wasp (Nw Zaland Chstnut Council Inc., 1997). Nw Zaland is on of th fw placs lft in th world, which is still fr of both ths psts. A divrs group of othr pathogns also affct chstnut (Macdonald, 1993). Ths includ bark disas' (causd by Cryphonctria parasitica Murr.), 'ink disas', (attributd to Phytophthora spp.). Basidiomyctous fungi that caus root dcay and powdry mildws. Numrous nvironmntal factors may dirctly influnc tr halth, but according to Macdonald (1993), most important is th rol thy play in prdisposing chstnut to a varity of biotic pathogns. Nw Zaland has a wid rang of fungal pathogns capabl of affcting chstnuts including Phomopsis, Botryospharia, Pnicillium, Asprgillus, Altrnaria, Fusarium, Botrytis, Rhizopus, Gotrichum, Pstalotia, Phoma, Trichodrma, Sclrotinia, Colltotrichum and Glomrlla spcis (Klinac and Forbs, 1995). Th nut rot associatd with Phomopsis castana causs srious losss to th Australian (Washington, Stwart-Wad and Hood, 1999) and Nw Zaland (Klinac, 1996) chstnut industris. It rducs storag lif, limits xport and markt potntial, and may caus production of th undsirabl mycotoxin 'phomopsin' (Klinac, 1996). A numbr of studis (Klinac, 1996; Washington and Stwart-Wad, 1996) hav attmptd to quantify th lvls of infction and dvlop control masurs by undrstanding th disas cycl of th causal fungus P. castana. Svral hundrd Phomopsis spcis nams appar in th litratur, many of which ar basd solly Agronomy N.Z. 3, 2 29

2 on host substrat (Brayford, 199). Phomopsis blongs to th Dutromycts (asxual fungi), in th ordr Spharopsidals (asxual spors producd in pycnidia) (Agrios, 1978). As was pointd out by Klinac and Forbs (1995), in Nw Zaland, thr ar fw data availabl that show how much of a problm nut fungal infctions ar. This, thy suggstd, was du to th problm of pllicl adhsion in th chstnut krnl making it difficult to assss mould infctions. Klinac and Forbs (1995) hav rportd, ovrsas, chstnut loss rats du to fungal infction of up to 82%, whil th mor typical avrag was btwn 13-25%. Th sam authors hav rportd that in Nw Zaland storag trials with Nw Zaland chstnut slctions, typical losss in 1993 wr btwn 2-44%, and in 1994 btwn 2-3%. Most of th Nw Zaland chstnut slctions xamind (12, 15 and 115), hav shown significant lvls of Phomopsis infction (Klinac, 1996). Howvr, Klinac (1997) notd that som noncommrcial Nw Zaland slctions wr quit rsistant to fungal infction. Stwart-Wad and Washington (1995) found that in Australia thr wr cultivars with low incidnc of Phomopsis and vry low natural rot lvls. Phomopsis was th most common fungus isolatd from insid th chstnut, but rarly from th. outsid, suggsting that infction occurs wll bfor harvst (Klinac, 1996). In an Australian study (Washington and Stwart-Wad, 1996), various parts of chstnut plants (including flowrs, lavs, shoots and oldr wood) wr tstd, with Phomopsis dtctd in most plant parts in all cultivars undr study. In that study summr fungicid sprays gav a dgr of control of rot dvlopmnt in nuts whil post-harvst fungicid dips did not markdly rduc rots in nuts during storag. Klinac (1996) found som vidnc in Nw Zaland for rgional diffrncs in Phomopsis infction. In Nw Zaland, in gnral, North Island orchards produc largr nuts, but with mor intrnal rot and surfac moulding. South Island orchards produc smallr nuts but with much lss fungal infction and rots (Gardnr t al., 1999). Bcaus chstnuts in Nw Zaland ar ssntially dstind for th xport markt, handling and storag conditions ar important (McNil and Gardnr, 1995). Th microflora of chstnut during storag and th postharvst tratmnts ar critical in dtrmining typs and lvls of storag rots, and vntually storag ability. In Australia, organisms isolatd most oftn from th shll or krnl tissu wr Phomopsis, Botrytis, unidntifid yast, Pnicillium, Cuthospora, unidntifid bactria, Cladosporium, Altrnaria, Truncat/la, Epicoccum and Phoma (Washington, Alin and Dooly, 1997). Klinac and Forbs (1995) hav pointd out that th storag of chstnuts is furthr complicatd by th gntics of chstnut itslf as ach nut is an out-frtilisd mbryo and gntically distinct from all othr chstnuts. Bcaus th nut rot associatd with P. castana is a major caus of loss during storag, this study was undrtakn to dtrmin th pidmiology of this fungus in chstnut trs. Matrials and mthods During th growing sason an xprimnt was carrid out to valuat th changs in P. castana infction lvls in diffrnt parts of th chstnut tr from mid Novmbr to th nd of April. Th monitoring of Phomopsis infction lvls was don on sampls collctd from 'Th 199 Lincoln Univrsity Chstnut Trial' stablishd on a Wakanui soil locatd at th Horticultur Rsarch Ara, Lincoln Univrsity, Cantrbury, Nw Zaland (latitud 43 39'S and longitud 1728'E) (Khan, McNil and Samad, 1998). Th trial was stablishd to tst yild and quality of a numbr of potntial commrcial lins. Full dtails of th managmnt for th first nin yars ar givn in McNil t al (2). Th trial was irrigatd by undr tr minisprinklrs as rquird to maintain >5% availabl soil watr capacity in th soil as indicatd by nutron prob radings takn on a 1-day basis. Wds wr controlld undr th trs by spraying with glyphosat as rquird. Howvr, th wds wr shadd out to a larg dgr by th trs. Th trs had bn prund th prvious wintr to rmov low branchs. Whil laf analyss suggstd no srious nutritional problms (McNil t al., 2), it was possibl that N, Ca, K and Mg may hav bn low. Hnc in 1998 th ara was surfac timd at 3tlha and frtilisr applications wr incrasd to 25g N, 1g P, loog K and 25g Mg pr tr dlivrd as a wintr sid drssing plus an additional 1g N pr tr dlivrd in th summr. Agronomy N.Z. 3, 2 3

3 This trial consistd of 17 cultivars with fiv rplications compltly randomisd. For th purpos of this xprimnt, six cultivars wr sampld, thr pollinator cultivars (Long Bay-4, Don Whlan and Crwnna-3) and thr main crop cultivars (15, 115 and 12). For ach cultivar, four randomly chosn rplicats (from 4 diffrnt trs) wr sampld. Twigs about 3-4 cm long, with lavs and flowrs wr cut and put in individual plastic bags for sam day laboratory procssing. All sampls wr rmovd from th tr up until th final sampl dat whn th nuts and burrs wr collctd from th ground. Isolation For ach twig, four stm disks wr cut at diffrnt positions along th twig. Th first thr stm disks wr obtaind from prvious sason's wood, whil th fourth on was obtaind from nw sason's wood. With a 7mm diamtr cork borr, two laf disks wr xtractd from th old lavs (thos that had formd at th start of th sason) and two laf disks from th young lavs (thos that wr rcntly fully xpandd). A 3-4 mm sampl from ach of four mal and fmal catkins (dividd into burrs and mbryo) was also obtaind. from ach twig whn availabl. All sampld disks from, lavs, flowrs and stm wr put into a nylon msh bag and strilisd by soaking for 3min in a 2.5% sodium hypochlorit (NaOCl) solution and thn rins-washd in stril distilld running watr for 5min. For th krnl and hypocotyl, th tripl strilisation procdur was 3s in 95% thanol, 3min in 2.5% NaOCl and 3 s in 95% thanol using th procdur dscribd by Johnson t al. (1991) and Andrson t al. (1997). Bark was dissctd from th stm and placd on th PDA sparatly. All sampls wr platd onto ptri plats containing potato dxtros agar (PDA, 39gllitr) undr stril Laminar Flow conditions and incubatd at 25"C undr a 12 h light/dark rgim. Tsting for Phomopsis Aftr 6 and 7 days, th plats wr xamind for fungal growth. For som plats th fungi provisionally idntifid as Phomopsis wr purifid and thir idntity confirmd by xamination of lactophnol staind culturs undr an Olympus light microscop. Fungi.45.4 't:j Q).s m.3 Cllr;:..21.1!! C. Cl).25 CI).!Q.2 :.15 a:.1 a..! t f I f! 6!! 5 c:=::j rainfall -o- oldstm 4 SE(man).. 3 iii a: 2 1 1/Nov/98 1/Dc/98 1/Jan/99 1/Fb/99 1/Mar/99 1/Apr/99 1/May/99 Dat of sampling Figur 1. Proportion of vgtativ sampls infctd Phomopsis castana throughout th 1998/99 growing sason. Th histogram bars indicat rainfall btwn sampl dats. Agronomy N.Z. 3, 2 31

4 wr idntifid on th basis of colony morphology and confirmd as Phomopsis. Raw data wr ntrd into an 'Excl' sprad sht and analysd by analysis of varianc (ANOV A) using th computr packag 'MINIT AB-11'. Rsults and Discussion Thr was a gnral dclin in th lvls of "dtctd" infction in all chstnut plant parts sampld from th start of th sason with th xcption of th old laf sampl on January 27th (Fig. 1). Infction lvls wr low in all sampls during th pak of th summr (January-Fbruary) whn tmpraturs and solar radiation wr high and rainfall low. All cultivars xamind showd Phomopsis infction, although ths lvls dclind ovr th priod of sampling up until th last fw sampling dats (Fig. 2). Howvr, thr wr cultivar diffrncs in both lvls of infction and in th rat of dclin of infction lvls (Figs. 2 and 3) with cv. Crwnna 3, Don Whlan and 115 bing particularly low. Klinac (1997) has suggstd that C. sativa chstnut typs wr gnrally mor havily infctd with fungi than C. crnata typs. Th data from this trial ar not ntirly consistnt with this assrtion as cv. Crwnna 3 and Don Whlan ar vry C. sativa lik in thir charactristics (Oraguzi and McNil, 1998). Howvr, th prsnc of Phomopsis alon could not b clarly corrlatd with chstnuts going rottn. A vry high lvl of ndmic Phomopsis infction xistd within th trial, particularly within th old bark of trs. Wintr isolations from th bark hav shown 1% infction of th trs tstd in this trial (McNil, prs.com. 1999). Howvr, in spit of th high ndmic lvls of Phomopsis, th fungus may only b consistntly isolatd from som locations of th tr. Th nw growth in particular (both vgtativ (Fig. 1) and rproductiv (Fig. 4)) showd rlativly littl infstation. Thr wr diffrncs in th lvls of Phomopsis infction btwn th slctions. This is in agrmnt with th obsrvations of Klinac (1996) who found no truly Phomopsis-fr or rsistant chstnut slction but a rang of lvls of infction, suggsting that som.3 "1:1.25 J!!.EgJ.2 lilt:.!!! l.1 t::o &.if a.. f! f f!!! t! r f 5 c:::::j rainfall 4 -o- SE (man) "E Crwnna 3 g --r- Long Bay 4 3 -o- Don Whlan jij c a a: 1/Nov/98 1/Dc/98 1/Jan/99 1/Fb/99 1/Mar/99 1/Apr/99 1/May/99 Dat of sampling Figur 2. Proportion of sampls infctd with Phomopsis castana in thr pollinator varitis (Crwnna 3.Lnn!l Rav 4. and Don Whlan). Rainfall btwn samnl dats is indicatd bv th bistmmtm bars Agronomy N.Z. 3, 2 32

5 slctions may b mor tolrant than othrs. In th prsnt xprimnt, two trs wr consistntly Phomopsis-fr during th ntir priod of th trial (tr no. 62 (cv. 115) and 3 (cv. Crwnna-3) data not shown), but, of cours, no gntic conclusion can b drawn from such obsrvation as othr trs of ths cultivars wr infstd. In a prliminary xprimnt, 99% of chstnut sdlings plantd out from bulk sd collctd from th trial wr infctd with Phomopsis. Th obsrvation that th bark was th main sourc of Phomopsis in sampls takn from th stm was in agrmnt with prvious rsults in anothr spcis, which indicatd a high frquncy of Phomopsis oblonga in lm outr bark (Wbbr and Gibbs, 1984). Ths rsults must b considrd as vry prliminary bcaus th wathr affcting th trial ara during th sampling priod was unusually dry. Wttr conditions may incras th lvl of fungal infction (Ogilvy, 1998). Moistur, lik tmpratur, influncs th initiation and dvlopmnt of infctious disass in many intrrlatd ways. Th most important influnc of moistur sms to b on th grmination of fungal spors and on th pntration of th host by th grm tub (Agrios, 1978) Prvious work (Klinac, 1996) has focusd mainly on th lvl of infction in th harvstd nut, which has bn found to b high (usually xcding 5%) crtainly much highr than th lvls found in this trial. At this stag, thr is no information on lvl of infction in th harvstd nuts aftr a priod of storag. Howvr, in a study by Wadia, Klinac and McNil (1999), it was found that th fungus P. castana was associatd with both rottn and halthy nuts in all th sampls, though thr was a larg variation in svrity of incidnc btwn cultivars as wll as locations. In th study, P. castana was found associatd with all th six cultivars from th Lincoln Univrsity orchard at vry low lvls. Th incras in lvls of infction dtctd in th mbryos and burrs on April 13 may corrspond to incrasd sugar lvls in th maturing nut, although it is mor likly rlatd to rainfall. It has bn suggstd that Phomopsis is isolatd mor frquntly from th hilum nd of th shll and krnl than from othr parts tstd (Washington t al, 1997). Phomopsis castana has l.3 "C.25 Q) jlb :Ei.2 UlCI) if 'E c. a.. I t f I f!! t! t f 5 6 c:::j rainfall 4 -o- SE(man) 12...,.._ o &! 1/Nov/96 1/Dc/96 1/Jan/99 1/Fb/99 1/Mar/99 1/Apr/99 1/May/99 Dat of sampling Figur 3. Proportion of sampls infctd with Phomosis castana in thr main crop varitis (12,15, and 115). Th histogram bars indicat rainfall btwn sampl dats. Agronomy N.Z. 3, 2 33

6 .45.4 j m! f I f f!! r!! r jj.3 _...catkin.5cil --- fmal flowr.25 mbryo Sl hilum nd C..-!!l E.2 _... mbryo-mid -- stylar nd burr a if SE(man).1 'E &. D.. 1/Nov/98 1/Dc/98 1/Jan/99 1/Fb/99 1/Mar/99 1/Apr/99 1/May/99 Dat of sampling Figur 4. Proportion of chstnut sampls from rproductiv organs infctd with Phomopsis castana throughout th growing sason. bn shown to b an ndophyt in chstnuts (Washington, Hood, Goubran, Hpworth, Stwart-Wad, 1998) and whil th mchanism of th dvlopmnt of th disas is not clar, it appars that th fungus coloniss nuts via th pduncl and hilum (Washington t al., 1998). According to Ogilvy (1998), sinc Phomopsis is an ndophyt rsiding in th vascular systm of th tr, it is unlikly that th major point of ntry of th infction is via th hilum. Howvr his obsrvation suggsts that Castana sativa is mor pron to infction this way than C. crnata or C. mollissima. Ogilvy (1998) blivd that thr is a continuing sourc of infction, th spors bing prsnt in most parts of th tr. Howvr, Rnhart (1999) found fungal infction (not conclusivly idntifid as Phomopsis) on th stigrnatic surfac suggsting infction via this rout rathr than via th hilum Sh also idntifid a trnd for lat pollinatd nuts to dvlop a highr incidnc of rot. Th data prsntd hr do not indicat any significant diffrnc btwn sampl locations within th nut (Fig. 4) with infction lvls in th nut always rmaining low and suggsting both routs of infction ar possibl. Th trial is bing continud to dtrmin what corrlation may xist btwn th infction lvls dtrmind during th vgtativ and flowring priod and Phomopsis-inducd rot in storag. Conclusion Furthr work in this ara should involv obsrvations ovr svral sasons to stablish th likly occurrnc of gnotyp by nvironmnt intractions. Howvr, at this stag, with appropriat cultivar slction, th Cantrbury climat appars to b poorly suitd to Phomopsis as a nut disas though it is wll suitd to Phomopsis as an ndophyt during wintr, arly spring and possibly lat autumn. Agronomy N.Z. 3, 2 34

7 Rfrncs Agrios, G.N Plant Pathology. 2nd d. Acadmic Prss, N.Y. p.73. Andrson, C.R., Johnson, G.I. and Gust, D.I Phonwpsis castana survivs as an ndophyt and a pathogn on th chstnut tr fruit Draft copy of a papr from th School of Botany, Univrsity of Mlbourn, Parkvill 352, Australia. Brayford, D Variation in Phonwpsis isolats from Ulnus spcis in th British Isls and Italy. MycologyRsarch 94(5), Gardnr, L., McNil, D.L., Savag, G. Kandula, W. and Klinac, D Nw Zaland chstnut nationwid quality survy. HortRsarch Clint Rport No.1999/214. Johnson, G.I., Mad, A.J., Cook, A.W. and Dan, J.R Mango stm and rot pathogns- infction lvls btwn flowring and harvst Annals of Applid Biology 119, Khan, Z.U., McNil, D.L. and Samad, A Root pruning rducs th vgtativ and rproductiv growth of appl trs growing undr an ultra high dnsity planting systm. Scintia Horticultura 77, Klinac, D.L. and Forbs, C Chstnut postharvst storag and handling. In Chstnut Growrs Handbook, Nw Zaland Chstnut Council. p Klinac, D.L Phonwpsis infction of Nw Zaland chstnuts. Rport to th Nw Zaland Chstnut Council. HortRsarch Clint Rport no.96/57. Klinac, D.L Idntification and control of fungal storag rots in Nw Zaland chstnut slctions. Rport to th Nw Zaland Chstnut CounciL HortRsarch Clint Rport no Macdonald, W.L Disass of chstnut Procdmgs of th Intrnational Congrss on Chstnut Spolto, Octobr 2-23,1993. McNil, D.L. and Gardnr, L Chstnut rsarch in th South Island, In Chstnut Growrs Handbook, Nw Zaland Chstnut Council. p McNil, D.L Postharvst handling of chstnuts in Nw Zaland, In Chstnut Cours, U.C. Davis, Octobr McNil, D.L., Gardnr, L., Null, C. and Mclntosh, K. 2. Rproductiv productivity and quality of chstnuts aftr yar 9 in a rplicatd varity trial in Cantrbury NZ. Southrn Nut Growrs Association: Halth in a Shll 39,4-11. Nw Zaland chstnut Council Chstnut in Cantrbury, A Growing Opportunity. Sminar Paprs Lincoln Univrsity 25 May Ogilvy, D Phonwpsis-whn dos it strik? Australian Nut Growr, Dcmbr 1998-Fbruary (4), Oraguzi, N.C., McNil, D.L., Klinac, D.L., Knowls, R.D. and Sdcol, J.R Rlationship of chstnut spcis and Nw Zaland chstnut slction using morpho-nut charactrs. Euphytica 99, Oraguzi, N.C. and McNil, D.L Congrunc btwn RAPD and Morpho-Nut markrs in rvaling gntic rlationships among NZ chstnut slctions. NZ Journal of Crop and Horticultural Scinc 26, Rnhart, C.M Chstnut Flowring and Pollination, A mastr Thsis. Waikato Univrsity. p.171. Stwart-Wad, S. and Washington, W.S. (Eds.) Th rot rports. Th chstnut growrs of Australia. Issu 1, Novmbr Wadia, K.D.R., Klinac, D. and McNil, D.L Survy of chstnut nut-rot in Nw Zaland. In Procdings of th 52nd N.Z. Plant Protction Confrnc. p Wright, W.R Storag dcays of domstically grown chstnuts. Plant Disas Rportr44(1l), Washington, W.S. and Stwart-Wad, S.M Phonwpsis nut rot of chstnuts-a rsarch updat. Australian Nut Growr, March Washington, W.S., Alin, A.D. and Dooly, L.B Prliminary studis on Phonwpsis castana and othr organisms associatd with halthy and rottd chstnut fruit in storag. Australasian Plant Pathology 26, Washington, W.S., Hood, V., Goubran, F., Hpworth, G. and Stwart-Wad, S Effct offungicids applid as foliar sprays and trunk injctions on nut rot of chstnuts causd by Phonwpsis castana in Victoria. Australian Journal of Exprimntal Agricultur 38, Washington, W.S., Stwart-Wad, S. and Hood, V Phonwpsis castana, a sd born ndophyt in chstnut trs. Australian Journal of Botany 47, Wbbr, J.B. and Gibbs, J.N Colonization of lm bark by Phonwpsis oblonga. Transactions of th British Mycological Socity 82(2), Agronomy N.Z. 3, 2 35