MANAGING BOTRYOSPHAERIA/PHOMOPSIS CANKERS AND ANTHRACNOSE BLIGHT OF WALNUT IN CALIFORNIA

Transcription

1 MANAGING BOTRYOSPHAERIA/PHOMOPSIS CANKERS AND ANTHRACNOSE BLIGHT OF WALNUT IN CALIFORNIA Themis J. Michailides, ShuaiFei Chen, David Morgan, Dan Felts, Mohamed Taieb Nouri, Ryan Puckett, Michael Luna, Janine Hasey, Kathleen Anderson, William Coates, Elizabeth Fichtner, Rick Buchner, and Walter Bentley ABSTRACT The Botryosphaeriaceae fungi and Phomopsis species are widely distributed in walnut orchards from almost all walnut-growing regions in California. Pathogenicity studies were done with 10 species of Botryosphaeriaceae and 2 species of Phomopsis, and we confirmed that these fungi can infect both shoots and fruit of walnut. We have now started studying the various factors that will influence spread and infection of these pathogens. We are also interested in exploring the possibility that these fungi can cause latent infections. As was done with the pistachio Botryosphaeria panicle and shoot blight disease, we will use ONFIT to determine whether latent infections occur, and use the BUDMON Technique to determine the incidence of these fungi in intact buds of walnuts. Both the conidial and the ascosporic stages of Botryosphaeriaceae and Diaportheceae (Phomopsis species) were found in walnut, suggesting that these fungal pathogens can spread by both splashing water and by air. In addition to Botryosphaeria and Phomopsis species, Fusarium spp., Alternaria alternata, Colletotrichum acutatum, Aspergillus niger, and a Gleosporium sp. can occur and decay walnut fruit in the field. Furthermore, lesions caused by the walnut blight pathogen, Xanthomonas arboricola pv. juglandis, can also be colonized by Botryosphaeriaceae fungi. We confirmed that walnut scales contribute to higher incidence of infection by Botryosphaeriaceae in comparison with the infection of shoots not bearing any scales. Although symptoms of Botryosphaeria canker and blight disease were not observed in 2013 during the growing season due to dry climatic conditions after harvest, we evaluated infections that started at harvest and postharvest on husks, peduncles, and shoots of walnuts. Interestingly, some of the registered fungicides that were applied in spring and early summer had a major effect in reducing infections of these tissues by Botryosphaeriaceae fungi. Although there was plenty of ascosporic inoculum of the anthracnose pathogen in walnut leaves dropped onto the orchard floor, the low precipitation in 2013 in the Hollister area resulted in no anthracnose development in this orchard where the fungicide trial was established. A proposed disease cycle of the walnut anthracnose is presented. OBJECTIVES 1) Diagnoses of diseased samples collected from fields and/or submitted by growers, farm advisors, and pest control advisers. 2) Survey systematically walnut orchards in three geographic areas (northern, central, & southern) for canker diseases and walnut scale infestation. 3) Determine and compare pathogenicity of the putative isolated pathogens. 4) Determine whether and how long major pruning wounds are susceptible to infection Botryosphaeriaceae fungi and Phomopsis species. 5) Determine putative association of walnut scale and infection by these fungal pathogens. 6) Understand the anthracnose disease cycle and develop ways to manage it. California Walnut Board 325 Walnut Research Reports 2013

2 PROCEDURES 1) Diagnoses of diseased samples collected from fields and/or submitted by growers, farm advisors, and pest control advisers. In the last decade or so, Botryosphaeria blights and cankers have caused major damage to major permanent crops in California, such as grapes, pistachios, and almonds. In addition, in the last few years, farm advisors have reported frequent killing of branches in English walnut very similar to branch wilt. Two groups of fungi, species of Botryosphaeriaceae and Phomopsis species were isolated from these branches instead of the fungal pathogen Hendersonula toruloidea that causes the branch wilt disease in walnut. A total of 74 walnut samples, such as tree trunks, scaffolds, shoots, fruit, and leaves showing putative infections were diagnosed in ) Survey systematically walnut orchards in three geographic areas (northern, central, & southern) for canker diseases and walnut scale infestation. Walnut samples were collected or sent to our laboratory representing 14 different counties. Examination of samples was done with a dissecting microscope and when needed a compound microscope to identify species of Botryosphaeriaceae or Phomopsis and other fungal species. The counties where samples were collected included Butte, Colusa, Fresno, Glenn, Kings, Merced, Placer, San Benito, San Joaquin, Stanislaus, Sutter, Tehama, Tulare, and Yolo. The distribution of 185 isolates kept in our fungal collection is shown in Figure 1. 3) Determine and compare pathogenicity of the putative isolated pathogens. Pathogenicity of fungi isolated from walnuts. Blighted fruit showing putative symptoms of fungal infections and sometimes fruit with typical walnut blight symptoms were collected and used in this study. The goals were to determine a) any putative fungi that infect and decay fruit, b) if fruit exhibiting symptoms of walnut blight can also be infected by Botryosphaeria, and c) any fungi that decay fruit on the trees and those fell on the ground. Isolations from tissues showing symptoms were done using acidified potato-dextrose agar and incubating the plates at 77 F (25 C). The fungi were identified to genus and then used in pathogenicity experiments. For the pathogenicity experiments, fruit of the cultivar Chandler were collected from the experimental walnut orchard at Kearney, surface sterilized in chlorinated water (10% bleach solution), excess water was dried under a positive flow hood, and the fruit were wounded and inoculated with a drop of a spore suspension of each putative pathogen. The inoculated fruit were incubated at 77 F (25 C) for 5 to 10 days, depending on the rate of growth of each putative pathogen. Determine incidence of Botryosphaeria spp. in walnut buds. Botryosphaeria spp. have been reported colonizing buds in pistachio and in fact, the incidence of bud colonization is used there to predict the presence and the severity of the blight in an orchard. To determine the incidence of Botryosphaeria spp. on buds of walnut, two cooperating farm advisors collected 50 random buds per orchard in four orchards in Tehama Co. and three orchards in Stanislaus Co. Cultivars sampled included, Howard, Chandler, Sexton, and Tulare. Buds were processed with the California Walnut Board 326 Walnut Research Reports 2013

3 BUDMON Protocol (APPENDIX I). Briefly, after surface sterilization the buds are plated (10 buds per plate) on the surface of acidified potato dextrose agar (APDA), incubated at 77-80ºF for about 5-7 days, and then the colonies of Botryosphaeriaceae are recorded. Determine the mode of infection of stems, branches, and fruit by Botryosphaeriaceae fungi. To determine when husks (hulls) are infected, first immature and then maturing fruit in the field were inoculated in the field with Lasiodiplodia citricola (isol. 6-I34) and Neofusicoccum parvum (isol. 1-L83), using 10 fruit per isolate. This was done by wounding the hulls with a 7-mm cork borer and placing a drop of a spore suspension ( conidia per ml ) or a mycelial plug into the wound. Water served as a negative control. Inoculations were done every 3 weeks, beginning May 16, 2013 with the last inoculation done September 20, Mycelium inoculations were done at the same time as the spore suspension inoculations and consisted of 7- mm PDA plugs from a 7-day-old culture. Hulls were wounded as above and agar plugs bearing mycelia placed upper surface down into the fresh wound. Ten hulls per isolate were inoculated. Ten additional hulls were wounded and inoculated with a sterile PDA plug and served as negative controls. Beginning July 16, 2013 spray inoculations were done at the same time as the other inoculations. Shoots with fruit were sprayed with the same spore suspension as used for the drop inoculation on wounded fruit, or water for the control treatment. The inoculations were performed in late afternoon, bagged with a plastic bag to maintain humidity, and covered in a white paper bag to prevent overheating. Bags were removed early the following morning. At harvest time, blighted fruit were recorded for each treatment. Some of the blighted fruit were then removed and isolations made from them to determine if the pathogen used for inoculation could be recovered. To determine when peduncles are infected, peduncles with the fruit removed were inoculated in the field with L. citricola (isol. 6-I34) and N. parvum (isol. 1-L83). This was done by carefully removing the fruit without breaking the peduncle and placing a 2 2 cm piece of cheesecloth saturated in a spore suspension over the wound. Suspensions of (some as low as ) conidia per ml were used. There were no control treatments. Inoculations were done weekly, with an additional inoculation done 3 days after the first inoculation, beginning September 20, 2013; the last inoculation was done on October 11, Empty husks were spray-inoculated in the field with the same suspensions used with the cheesecloth method. This began on October 11, 2013 and was repeated weekly with an additional inoculation done on October 14, The last inoculation was done on November 1, There were no control treatments used. Both of the groups are currently being monitored, with photographs taken to document lesion development over time. Cankers will be recorded in spring of Empty husks were collected after harvest and inoculated in the laboratory with the same isolates of both pathogens and evaluated for the development of pycnidia. Hulls were dipped for three minutes in either water or a 10% bleach solution containing 0.5 ml Tween-20 per liter. They were placed in plastic containers over plastic racks, spread so there were not in contact with each other, and inoculated with a 7 mm agar plug with N. parvum or L. citricola. They were incubated at 25 C, then rated from 0 to 4 for presence of pycnidia, with 0 being no pycnidia and California Walnut Board 327 Walnut Research Reports 2013

4 4 being mostly covered with pycnidia. The first inoculation was done on November 14, 2013 and recorded after 11 days. The second was done on November 24, and recorded after 15 days. Inoculations with other fungi isolated from blighted walnut fruit. Because three other fungi (in addition to Phomopsis and Botryosphaeria species) were frequently isolated from blighted walnuts, we wanted to determine whether they can decay walnut fruit and compare them with the decay caused by Phomopsis and Botryosphaeria spp. Walnut fruit of the cultivar Vina were used for these inoculations. Fruit were inoculated in the Kearney orchard on July 7 and again on August 16, Fruit were wounded with a 7- mm cork borer and the plug removed from the hull. A 7-mm agar plug with mycelium was placed in each well created with the cork borer and then covered with a piece of a white label tape. Agar plugs with no mycelia were used as control treatments. The isolates used for these inoculations were Phomopsis (isol. 8-D14), Botryosphaeria dothidea (isol. 8-D15), Colletotrichum acutatum (isol. 8-D11), (Gloeosporium species with Marssonina type conidia; isol. 8-D12), and a Fusarium species (isol. 8-D10). At harvest time (October 21, 2013), blighted fruit were recorded for each treatment. Some of the blighted fruit were then removed and isolations made from them to determine if the pathogen used for inoculation could be recovered. 4) Determine whether and how long major pruning wounds are susceptible to infection Botryosphaeriaceae fungi and Phomopsis species. Due to the postdoc Dr. ShuaiFei Chen leaving for another position after completing his 1-year contract, this experiment was not initiated; it will be initiated this winter and fall of ) Determine putative association of walnut scales and infection by Botryosphaeriaceae fungi. Because walnut scales were again common in many of the blighted branches, we wanted to confirm last year s experiments in which walnut scales resulted in higher incidence and more severe infection by Botryosphaeriaceae. Ten 1-year old shoots bearing scales and ten 1-year shoots without scales each were inoculated with Lasiodiplodia citricola (isol. 6-I34) or Neofusicoccum parvum (isol. 1-L83). Inoculations were performed either by placing a 7-mm mycelial plug or a 40 µl drop of a 20,000 spores/ml suspension in the middle of the 25 cm shoots. The inoculated shoots were incubated at 77ºF for 2 months and then the incidence of infection was recorded. Fungicide trials to control Botryosphaeria/Phomopsis canker and blight. Tulare Co. trial: We established a fungicide trial in Tulare Co. in cooperation with farm advisor Elizabeth Fichtner. The walnut trees (cv. Chico) are spread randomly (trees from an abandoned orchard) in a 50-acre golf course close to Woodlake town. Blighted shoots collected from these trees indicated a 100% occurrence of Botryosphaeria species. The names of the fungicides and their label rates used in this trial are shown in Table 5. Due to dry weather no disease developed at harvest time. However, we plan to visit and evaluate this trial in early spring to determine whether any cankers that started developing in the fall, will increase in size by the spring California Walnut Board 328 Walnut Research Reports 2013

5 Because this trial represents an old cultivar and the trees are not cultivated under commercial cultural practices, this block will not be used again in future trials. Instead, another orchard where commercial cultural practices are used will be chosen for the fungicide efficacy trial. Butte Co. trial: This trial was set in cooperation with PCA Cliff Kitayama (Chico, CA) in a commercial orchard close to the town of Durham. The fungicides used were Abound, Quadris Top, Quilt Excel, Pristine, Luna, Experience, Luna Sensation, and Fontelis at label rates (Table 6). Five-acre plots with 5 acres buffers and 5-acre untreated controls were used for each treatment. The sprays were applied on May 16, mid June, and mid July. Due to dry weather no disease developed in green tissues at harvest. The orchard was checked once about 2 weeks after harvest, and again on October 25, when we noticed infections that moved into the peduncles, and some into the sustaining shoots. One hundred peduncles, shoots, and husks were collected from each treatment plot to determine the levels of Botryosphaeriaceae and any possible effect of the fungicide sprays. Results are presented as the percent of plated tissues produced Botryosphaeriaceae fungi in agar media. The flags of these plots will be kept until next the spring of 2014 to monitor the advancement of cankers. Samples of 100 buds will be collected from each treatment plot in late March 2014 and processed with the BUDMON technique (APPENDIX I) to determine the long effect of fungicides in infestation of buds by species of Botryosphaeriaceae. Tehama Co. trial: This trial was set in cooperation with a grower in a commercial orchard in Tehama Co. The fungicides used were Fontelis, Pristine, Luna Experience, Luna Sensation, Quash, Bumper, Abound, Quadris Top, Quilt Excel, and Kocide 2000, all at label rates (Table 6). Five-acre plots with row buffers and 5- acre untreated controls were used for each treatment. The sprays were applied on May 21, June 25, and August 14. Again, due to dry weather, no disease developed in green tissues at harvest. After harvest on October 25, 100 peduncles, shoots, and husks were collected from each treatment plot to determine the levels of Botryosphaeriaceae and any possible effect of the fungicide sprays. Results are presented as the percent of plated tissues (10 pieces of tissue per Petri plate containing acidified PDA) produced Botryosphaeriaceae fungi in agar media. The flags of these plots will be kept until the spring of 2014 to monitor the advancement of cankers and probably. Samples of 100 buds will be collected from each treatment plot in late March 2014 and processed as above with the BUDMON technique to determine the long effect of fungicides in infestation of buds by Botryosphaeriaceae spp. 6) Understand the anthracnose disease cycle and develop ways to manage it. Walnut anthracnose disease, caused by the fungus, Gnomonia leptostyla (the imperfect stage of the fungus Marssonina juglandis) has created some problems in walnut cultivars in commercial orchards and walnut rootstock plants (i.e., black walnut) in nurseries. Because the anthracnose disease was severe in the San Benito orchard in 2012 and we found both the conidia and the ascosporic stages of the fungus in this orchard, with the cooperation of Farm Advisor Bill Coates, we established a fungicide efficacy trial. The fungicides used and the label rates are listed in Table 7. There were five replicated trees per fungicide treatment in this trial. California Walnut Board 329 Walnut Research Reports 2013

6 RESULTS AND DISCUSSION 1) Diagnoses of diseased samples collected from fields and/or submitted by growers, farm advisors, and pest control advisers. We diagnosed 74 walnut samples in With the exception of 12 samples, all the rest produced mainly species of Botryosphaeria and/or Phomopsis. The majority of these samples had pycnidia of these fungi. Pycnidia are fruiting structrures produced in infected plant tissues underneath the epidermis layer producing pycnidiospores. Pycnidiospores require water to exude from the pycnidia and spread to infect plant tissues. In four of these samples that involved blighted shoots of at least 1 year old shoots (3 from Butte Co and one from Tulare Co.), in addition to the pycnidia of Botryosphaeria and Phomopsis, we found also perithecia produced by these fungi. Perithecia are fruiting structures that produce asci (little sacks) with 8 ascospores each. The importance of this type of spores is that when the perithecia get wet (i.e. from rains or sprinkler irrigation), the ascospores are forcibly ejected into the air and become airborne. In previous studies we also determined perithecia in walnut branches collected from the ground in commercial orchards in Stanislaus, Butte, Colusa, and Sutter Counties. The perfect state of Phomopsis species belongs in the genus Diaporthe. Two species of Diaporthe (D. rhushicola and D. neotheicola) isolated from walnuts were identified (Chen et al., 2014). Therefore, both species of Botryosphaeriaceae and the Phomopsis species produce both pycnidiospores and ascospores in walnut. The diagnosis of each sample was communicated on time to farm advisors, pestcontrol advisers, and growers who sumbitted samples in ) Survey systematically walnut orchards in three geographic areas (northern, central, & southern) for canker diseases and walnut scale infestation (distribution of pathogens). The counties where samples were collected include Butte, Colusa, Fresno, Glenn, Kings, Merced, Placer, San Benito, San Joaquin, Stanislaus, Sutter, Tehama, Tulare, and Yolo. Ten species of Botryosphaeriaceae and two species of Diapotheciae (Phomopsis spp.) were identified based on molecular and morphological characteristics, and these species were reported in last year s walnut research report. Among these species, Neofusicoccum mediterraneum is dominant, with 76% of the isolates were identified as this fungus. Neofusicoccum mediterraneum was widely distributed and was recovered from 10 different counties (Figure 1). In fact, this species was found to be the most common among isolates recovered from Botryosphaeria panicle and shoot blight of pistachio in California. In contrast to the California situation, two random isolates from walnuts in Greece were identified as Botryosphaeria dothidea and among 11 walnut isolates from blighted walnuts in Spain, eight were identified as Neofusicoccum parvum, 1 as Diplodia mutila, and 2 Phomopsis species. It is worth mentioning that the Botryosphaeria/Phomopsis canker and blight has caused significant damage to walnuts in Spain. Although the California map shows the distribution of isolates recovered and stored in our collection until early 2013, we also isolated isolates of Diaporthe species (Phomopsis species) from all these counties where Botryosphaeriaceae were isolated, but not all these isolates were stored in our collection. These results indicate that both the Botryosphaeriaceae and the Diaportheceae fungal pathogens are widespread among the walnut groves and the inoculum of these pathogens is very abundant throughout California. Because these fungi require water to California Walnut Board 330 Walnut Research Reports 2013

7 spread and cause infection, the dry conditions in 2012 and 2013 prevented the development of active infections of green tissues during the season. However, infections were observed at harvest and postharvest. 3) Determine and compare pathogenicity of and mode of infection by the putative isolated pathogens. a) Determine any possible fungi attacking walnut fruit. Inoculations of Chandler fruit with the three most commonly isolated fungi (Colletotrichum, Fusarium, and Gleosporium species) resulted in decaying the husks of fruit, and in some instances invading the kernel of the nuts. After 3 weeks incubation at 77ºF, Colletotrichum acutatum developed an average of 22 mm lesion, an isolate of Fusarium sp. developed a lesion of 15 mm, and a Gleosporium sp. a lesion of 16 mm. These results indicate that in addition, to Botryosphaeriaceae and Phomopsis spp., other fungal species can decay fruit in walnut orchards. Another fungus that was also commonly isolated from husks and kernels of walnuts was Alternaria alternata. However, pathogenicity studies (Koch s postulates) with this species will be completed in b) Determine whether fruit with symptoms of walnut blight can be infected by Botryosphaeria fungi. Because fruit with walnut blight also showed infections by a species of Botyosphaeriaceae, we inoculated fruit with an isolate of Botryosphaeria dothidea. After incubation for 2 weeks, 100% of the fruit had developed lesions of Botryosphaeria with 75% of them had exuding pycnidia on top of the walnut blight lesions. The results suggest that walnut blight lesions can eventually be colonized by Botryosphaeria which can produce abundant pycnidia to provide spore inoculum in the orchard. This experiment was repeated once with similar results. These results suggest that in walnut orchards, the bacterial disease walnut blight can initiate infections entering through the stylar end and create stylar end necrosis and at least the Botryosphaeriaceae may follow these types of infection. In Italy, however, Belisario et al. (2002) only occasionally isolated the walnut blight pathogen, Xanthomonas arboricola pv. juglandis, from brown apical necrosis caused by Fusarium, Alternaria, Cladosporium, Colletotrichum, and Phomopsis species. c) Determine differences of infection by fungi of fruit collected from the tree and the ground. Several fungi were isolated from blighted fruit from either fruit collected directly from the trees and the ground. The incidence of the fungi varied based on the orchard and whether the fruit was collected from the tree or the ground. The most common fungi isolated were Botryosphaeria or Phomopsis, Fusarium, and Alternaria alternata. In two of the orchards, Aspergillus niger was also common in nuts collected from the tree and from the ground (Table 1). d) Botryosphaeriaceae and Phomopsis spp. in walnut buds. The results of plating the walnut buds from orchards in Tehama and Stanislaus Counties in media and the recovery of Botryosphaeria spp. are shown in Tables 2 and 3. Similarly high levels of incidence of Botryosphaeria were recovered from buds in the sampled orchard in either County (Tehama or Stanislaus). In general, male buds in all the sampled orchards had higher incidence of Botryosphaeria spp. probably because these buds are bulkier than the female buds and represent more plant tissues plated in the agar media in comparison with the tissue of the female buds. California Walnut Board 331 Walnut Research Reports 2013

8 4) Determine whether and how long major pruning wounds are susceptible to infection Botryosphaeriaceae fungi and Phomopsis species. This experiment will initiate in ) Determine putative association of walnut scales and infection by these fungal pathogens. For the plug inoculations, the incidence of infection of shoots bearing walnut scales was higher than that of shoots without scales for either species inoculated (Table 4). For the spore suspension inoculation, only the inoculations with Lasiodiplodia citricola had higher infection rate on shoots with scales in comparison with the incidence of infection of shoots without walnut scales. For the Neofusicoccum parvum inoculations, both the shoots with and those without scales had the same infection rates. The results confirmed last year s results. All infected shoots were covered with pycnidia even after 1 month of incubation. Scales have been reported to predispose beech trees to canker diseases (Hale et al., 2006; Holmes, 1978; McCullough et al., 2005; Vance 1995), and it seems that is the case in walnuts as well (Michailides, et al., Walnut Research Reports 2012). Determine efficacy of fungicides to control Botryosphaeria and anthracnose of walnut. Tulare Co. trial: Disease did not develop in this trial (Table 5). On September 17, when the treatments were evaluated we found no infection of green tissues. However, we plan to revisit this orchard in spring 2014 and determine if any of the fungicide treatments had any effect in the canker development during fall and during the postharvest phase of the disease. Butte Co. trial: In this trial, there was a major effect of the fungicides applied in the spring in reducing the incidence of Botryosphaeriaceae in shoots and husks of treated walnut trees in compariosn with those of the untreated control trees (Figure 2). The effects on the incidence of infected peduncles varied. This may be because the level of peduncle infection was lower as compared to the levels of infected shoots and husks by Botryosphaeriaceae, which were more than four times greater than the infection of peduncles (Figure 2D). Tehama Co. trial: In this trial, the effects of the spring/summer fungicide sprays in reducing the incidence of Botryosphaeriaceae in shoots, peduncles, and husks were very variable, although some of the fungicide treatments (i.e. Pristine, Quilt Xcel, Quadris Top, Tilt, and Luna Experience, etc.) were effective in reducing Botryosphaeriaceae (Figure 3). The levels of infection of all shoots, peduncles, and husks of untreated control trees in this block were lower in comparison with those in the Butte Co. block. Again, the peduncles from the untreated trees had lower rate of infection in comparion with the infection rates of shoots and husks (Figure 3D). There are no statistics in either of these commercial fungicide trials because the plots were not replicated. However, each treatment represents a 5-acre plot separated with 5-acre buffer trees and the incidence of infection was based on 100 randomly collected shoots, peduncles, and husks. However, these results suggest that spring and summer sprays will have efficacy against the Botryosphaeria canker and blight disease and should be considered in future trials. Furthermore, the results suggest infections can develop during harvest time and after harvest, California Walnut Board 332 Walnut Research Reports 2013

9 depending on weather conditions, and thus these infections need to be monitored 2 months after harvest, and /or in the next spring. If there is a significant increase of the cankers size during this period, this will imply that in years with dry weather, infections by Botryosphaeriaceae may develop during fall. This contention is supported by the fact that we observed in many cases infections that moved though the peduncle into the sustaining shoots by October 25, ) Understand the anthracnose disease cycle and develop ways to manage it. Based on observations made in 2012 when the anthracnose disease was severe in the walnut orchard in San Benito Co., and after examining leaves collected from the orchard ground in March 2012 and 2013 we now can propose a disease cycle that applies for the walnut anthracnose under California conditions. Perithecia of the pathogen were found along the mid rib and side veins of leaves containing the characteristic ascospores of Gnomonia leptostyla, the perfect stage of the anthracnose pathogen Marssonina juglandis (Figure 4). Presumably, the ascospores produced in the perithecia served as the primary inoculum for infection of emerging leaves in the spring. That is the reason why the first fungicide spray should be applied in the spring (early to mid April) as leaves emerge and become about the ½ of their final size, This sprays protect the leaves from primary infections by ascospores. However, in order for the ascospores to become airborne, perithecia need to become wet by a rain. It is expected that sprays applied before a rain event during early season should be very effective in controlling infections as it has been shown in other similar diseases. The first efficacy trial in 2012 showed that several fungicides (Inspire Super, Luna Experience, Pristine, and Quilt Xcel) that are registered in walnut showed excellent efficacy against the anthracnose disease of leaves and fruit when they were applied at the right time (first spray when leaves were ½ of the final stage). In 2012, an average of 70% of leaves and fruit were infected. A larger trial was established in the same orchard in 2013 and was evaluated on August 13. No disease developed in 2013 (Table 7). A comparison of the rain pattern from March 20 to September 30 and the total precipitation during 2012 and 2013 can explain the lack of disease in Precipitation during the growing season in 2013 was 13 times less than tat in 2012 (Table 8). The San Benito plot will again be used to manage anthracnose since this is the location where anthracnose can develop under the right conditions. Weather data will be retrieved for 2011, 2012, 2013, and 2014 to determine rain patterns that are associated with disease development. Weather data will be checked for correlations with disease incidence during this period of time. If anthracnose does not develop, the trial will be evaluated for Botryosphaeria blight. Apparently, copper fungicides and Mannex that are applied against walnut blight cannot control anthracnose and that is the reason why research is needed to develop control measures for this disease with other fungicides registered in walnuts. There are other fungicides that are effective in controlling this disease as it has been reported in other States, but no studies were ever performed in California against anthracnose until in In 2012, we sampled and surveyed several orchards for anthracnose and we found that the disease is not only a problem of black walnut but also several cultivars of the English walnut can be severely infected. These include but are not limited to Ashley, Chandler, Hartley, Serr, Payne, Livermore, Tulare, and Mountner. A comparative evaluation of various cultivars done by the farm advisor William Coates (UCCE San Benito Co.) suggested that symptoms were the least severe in Howard and Tulare, the most severe in Serr and Payne, while Hartley and Chandler showed intermediate severity symptoms (Coates, 2012 Annual Walnut Report). California Walnut Board 333 Walnut Research Reports 2013

10 CONCLUSIONS 1) The majority of samples of blighted shoots collected or brought to our laboratory were diagnosed with infections by members of the Botryosphaeriaceae and Diaportheceae (Phomopsis species). 2) Botryosphaeriaceae and species of Phomopsis were isolated from samples from 14 counties in California, suggesting that these fungi are widely distributed in walnut orchards. 3) Both the water splashed (pycnidiospores) and the airborne (ascospores) inocula were discovered in walnut orchards in several counties. 4) In addition to Botryosphaeriaceae and species of Phomopsis, three other fungi, Colletotrichum acutatum, Fusarium, and Gleosporium species can also decay walnuts in the orchard. 5) Walnut fruit infected by walnut blight can also be colonized by Botryosphaeriaceae fungi, species of Phomopsis, Fusarium, Gleosporium, Aspergillus niger, and Alternaria alternata. 6) Male and female buds of walnuts can be infested/infected by Botryosphaeriaceae fungi; the incidence of these fungi in buds may serve as an indication of the inoculum levels in a walnut orchard. 7) For a second year, walnut scales contributed to higher levels of infection of shoots by Botryosphaeriaceae fungi than those of shoots without scales. 8) Botryosphaeria canker and blight disease seems to have a postharvest phase (infection and development of blights at harvest and postharvest). 9) In at least three orchards, we observed infections of husks, peduncles, and the sustaining shoots after harvest, although only occasional fruit blight was observed before harvest. 10) Some fungicides applied in the spring and summer reduced the postharvest infections of husks, peduncles, and shoots by Botryosphaeriaceae species as determined by plating these tissues in agar media. 11) Both the water splashed and airborne spore inocula of the walnut anthracnose pathogen were discovered in a walnut orchard, which is an indication that the pathogen can spread with splashing rain and by air. 12) The walnut anthracnose pathogen can cause secondary infections in English walnut orchards. 13) The airborne ascosporic stage of the anthracnose disease pathogen was found only on overwintered leaves on the orchard floor while conidia were observed on leaf, petiole, fruit, and shoot lesions on the tree and the ground. 14) The very low precipitation in 2013 in the Hollister area resulted in no disease development in 2013 in the orchard where the fungicide trial was established. 15) A disease cycle for the walnut anthracnose is proposed based on field observations. California Walnut Board 334 Walnut Research Reports 2013

11 RELEVANT LITERATURE Chen, S.-F., Morgan, D. P., Hasey, J. K., Anderson, K., and Michailides, T. J Phylogeny, morphology, distribution, and pathogenicity of Botryosphaeriaceae from English walnut in California. Plant Disease (in press): Belisario, A., M. Maccaroni, and L. Corazza Occurrence and etiology of brown apical necrosis on Persian (English) walnut fruit. Plant Disease 86: Coates, W. W Varietal susceptibility of English walnuts to walnut anthracnose disease (Ophiognomonia leptostyla). Walnut Research Reports 2012, pp Michailides, T. J., and Z. Ma Effects of nutritional and water stress on Botryosphaeria blight of pistachio. Pistachio Commission Production Research. Report, Crop Year 1999, pp Michailides, T. J., Z. Ma, D. P. Morgan, and D. Felts Effects of water and nutritional stress on Botryosphaeria panicle and shoot blight of pistachio. Pistachio Commission Production Research. Report, Crop Year 2000, pp Hale, F., Wiggins, G., Lambdin, P., and Grant, J Beech scale, a potential threat in the landscape. University of Tennessee, Extension Bulletin SP503-H. 3pp. Holmes, F. W Canker diseases of trees and shrubs. J. of Arboriculture 4(2): McCullough, D. G., Heyd, R. L., O Brien, J. G Biology and management of beech bark disease. Extension Bulletin E-2746, Michigan State University Extension. 12 pp. Ma, Z., Morgan, D. P., and Michailides, T. J Effects of water stress on Botryosphaeria blight of pistachio caused by Botryosphaeria dothidea. Plant Disease 85: Michailides, T. J., S. Chen, W. Coates, D. Morgan, R. Puckett, J. Hasey, K. Anderson, R. Buchner, C. DeBuse, E. Fichtner, and W. Bentley Managing anthracnose blight and Botryosphaeria and Phompsis cankers of walnut. Walnut Research Reports 2012, California Walnut Board, Folsom, CA. pp Vance, R. A., Incidence and life history of beech scale, initiator of beech bark disease, in the Great Smoky Mountains National Park. M.S. Thesis, University of Tennessee, Knoxville, 72 pp. California Walnut Board 335 Walnut Research Reports 2013

12 Table 1. Incidence of putative pathogens isolated from blighted fruit collected from the trees or the ground in various orchards (#1 to #3 in Tehama Co. and #4 in Sutter Co.) collected in mid July 2013 (#4) and on Sept 9, 2013 (#1 to #3). Isolations were made directly from the inner surface of the infected tissues by plating small pieces on the surface of acidified potato dextrose agar (APDA). Orchard Fruit collection site Walnut blight Botryosphae ria /Phomopsis (%) Fusarium (%) Alternaria alternata (%) Aspergillus niger (%) Gleospo rium (%) 1 Tree Tree Tree Tree ND Ground Ground ND Table 2. Incidence of Botryosphaeriaceae species recovered from flower and vegetative buds of various walnut cultivars in Tehama County (c/o Rick Buchner). Type of buds Incidence of Botryosphaeriaceae species (%) Orchard #1 (Howard) Orchard #2 (Howard) Orchard # 3 (Chandler) Orchard #4 (Howard) Male buds Female/Vegetative buds California Walnut Board 336 Walnut Research Reports 2013

13 Table 3. Incidence of Botryosphaeriaceae species recovered from flower and vegetative buds of various walnut cultivars in Stanislaus County (c/o Kathleen Anderson). Type of buds Incidence of Botryosphaeriaceae species (%) Orchard #1 (Sexton) Orchard # 2 (Chandler) Orchard #3 (Tulare) Male buds 50* Female/Vegetative buds Table 4. Effect of walnut scales on infection of shoots by Botryosphaeriaceae species. Fungal species used as inoculum 1 Walnut scales presence/ absence Incidence of infection (%) 7-mm mycelial plug inoculum Lasiodiplodia citricola (isol. 6-I34) + 70 Lasiodiplodia citricola (isol. 6-I34) 60 Neofusicoccum parvum (isol. 1-L83) + 40 Neofusicoccum parvum (isol. 1-L83 10 >20,000 spores/ml spore suspension Lasiodiplodia citricola (isol. 6-I34) + 30 Lasiodiplodia citricola (isol. 6-I34) 10 Neofusicoccum parvum (isol. 1-L83) + 30 Neofusicoccum parvum (isol. 1-L Ten 1-year old shoots were inoculated without wound in the center of a 25 cm shoot, incubated at 77ºF for 2 months and then recorded. California Walnut Board 337 Walnut Research Reports 2013

14 Table 5. Efficacy of fungicides against Botryosphaeria blight of walnuts (cv. Chico) in Woodlake, Tulare County in U p to 100 leaves and fruit for each of 5 replicated trees were recorded on September 17, Numbers followed by different letters are significantly different according to the LSD test at P = Statistical analysis was performed on arcsine transformed data. 3 The surfactant Latron 1956 at 0.06% vol./vol. was added. Treatment (fungicide(s)) Application dates Rate April `1 May 10 July 11 Fruit with lesions 1 (%) Fontelis 20 fl oz Fontelis Fontelis Fontelis 0 a 2 0 a YT fl oz YT669 YT669 YT669 0 a 0 a Fontelis+ 14 fl oz Fontelis+ Fontelis+ Fontelis+ 0 a 0 a YT669 6 fl oz YT669 YT669 YT669 Merivon oz/a + Merivon Merivon Merivon 0 a 0 a Merivon oz/a Merivon Merivon Merivon 0 a 0 a Merivon oz/a Merivon Merivon Merivon 0 a 0 a Pristine oz Pristine Pristine Pristine 0 a 0 a Quadris Top 3 14 Quadris Quadris Quadris 0 a 0 a Viathon 32 fl oz Viathon Viathon Viathon 0 a 0 a Viathon 48 fl oz Viathon Viathon Viathon 0 a 0 a Luna Luna Exp Luna Exp Luna Exp 0 a 0 a 6 fl oz Experience Luna Luna Exp Luna Exp Luna Exp 0 a 0 a 8 fl oz Experience Luna Luna Sen Luna Sen Luna Sen 0 a 0 a 5 fl oz Sensation K Phite 7LP 3 quarts K Phite K Phite K Phite 0 a 0 a K Phite 7LP + 3 quarts K Phite + K Phite + K Phite + 0 a 0 a DKP XTRA 2 quarts DKP DKP DKP Badge X2+ 4 lbs Badge + Badge + Badge + 0 a 0 a Manzate prostick) 2.4 lbs Manzate Manzate Manzate S oz S-2200 S-2200 S a 0 a Quash 3.5 oz Quash Quash Quash 0 a 0 a S oz S-2200 S-2200 S a 0 a Quash 3.5 oz Quash Quash Quash Regalia 2 qts Regalia Regalia Regalia 0 a 0 a Regalia + 1 qts Regalia + Regalia + Regalia + 0 a 0 a Quadris Top 14 fl oz Quadris Quadris Quadris Regalia + 2 qts Regalia + Regalia + Regalia + 0 a 0 a Quadris Top 14 fl oz Quadris Quadris Quadris Control Untreated 0 a 0 a Peduncles with lesions 1 (%) 1 California Walnut Board 338 Walnut Research Reports 2013

15 Table 6. Fungicides and rates per acre used in the trials to control Botryosphaeria/Phomopsis canker and blight of walnut in two walnut orchards, one each in Butte and Tehama Counties. Fungicides in Butte Co. trial: Active ingredient Rate per acre Fontelis 20.4% penthiopyrad + R oz Pristine 12.8% pyraclostrobin % boscalid oz R-11 Luna Experience 17.6% fluopyram % tebuconazole 9.6 fl oz Luna Sensation 17.6% fluopyram % trifloxystrobin 7.6 fl oz Abound 22.9% azoxystrobin 12.0 fl oz Quadris Top 18.2% azoxystrobin % 14.0 fl oz difenconizole Quilt Xcel 13.5% azoxystrobin % 21 fl oz propiconazole Untreated Fungicides in Tehama Co. trial: Active ingredient Rate per acre Fontelis 20.4% penthiopyrad + R oz Pristine 12.8% pyraclostrobin % boscalid oz R-11 Luna Experience 17.6% fluopyram % tebuconazole 9.6 fl oz Luna Sensation 17.6% fluopyram % trifloxystrobin 7.6 fl oz Quash 50% metconazole 3.5 oz Bumper 41.8% propiconazole 8 fl oz Quadris Top 18.2% azoxystrobin % 14.0 fl oz difenconizole Quilt Xcel 13.5% azoxystrobin % 21 fl oz propiconazole Kocide % copper hydroxide 6-9 lbs Untreated California Walnut Board 339 Walnut Research Reports 2013

16 Table 7. Efficacy of fungicides against anthracnose of walnuts (Marssonina juglandis) in San Benito County in Treatment (fungicide(s)) Application dates Rate April 9 May 17 July 1 Fruit with lesions 1 (%) Leaves with lesions 1 (%) 1 Fontelis 20 fl oz Fontelis Fontelis Fontelis 0 a 2 0 a YT fl oz YT669 YT669 YT669 0 a 0 a Fontelis+ 14 fl oz Fontelis+ Fontelis+ Fontelis+ 0 a 0 a YT669 6 fl oz YT669 YT669 YT669 Merivon oz/a + Merivon Merivon Merivon 0 a 0 a Merivon oz/a Merivon Merivon Merivon 0 a 0 a Merivon oz/a Merivon Merivon Merivon 0 a 0 a Pristine oz Pristine Pristine Pristine 0 a 0 a Quadris Top 3 14 Quadris Quadris Quadris 0 a 0 a Viathon 32 fl oz Viathon Viathon Viathon 0 a 0 a Viathon 48 fl oz Viathon Viathon Viathon 0 a 0 a Luna Luna Exp Luna Exp Luna Exp 0 a 0 a 6 fl oz Experience Luna Luna Exp Luna Exp Luna Exp 0 a 0 a 8 fl oz Experience Luna Luna Sen Luna Sen Luna Sen 0 a 0 a 5 fl oz Sensation K Phite 7LP 3 quarts K Phite K Phite K Phite 0 a 0 a K Phite 7LP + 3 quarts K Phite + K Phite + K Phite + 0 a 0 a DKP XTRA 2 quarts DKP DKP DKP Badge X2+ 4 lbs Badge + Badge + Badge + 0 a 0 a Manzate prostick) 2.4 lbs Manzate Manzate Manzate S oz S-2200 S-2200 S a 0 a Quash 3.5 oz Quash Quash Quash 0 a 0 a S oz S-2200 S-2200 S a 0 a Quash 3.5 oz Quash Quash Quash Regalia 2 qts Regalia Regalia Regalia 0 a 0 a Regalia + 1 qts Regalia + Regalia + Regalia + 0 a 0 a Quadris Top 14 fl oz Quadris Quadris Quadris Regalia + 2 qts Regalia + Regalia + Regalia + 0 a 0 a Quadris Top 14 fl oz Quadris Quadris Quadris Control Untreated 0 a 0 a leaves and fruit for each of 5 replicated trees were recorded on August 13, Numbers followed by different letters are significantly different according to the LSD test at P = Statistical analysis was performed on arcsine transformed data. 3 The surfactant Latron 1956 at 0.06% vol./vol. was added. California Walnut Board 340 Walnut Research Reports 2013

17 Table 8. Precipitation during the growing season and total precipitation in Hollister, San Benito in 2012 and Period March 20 to September mm 10.6 mm Total 425 mm 133 mm Anthracnose incidence 70% 0 % California Walnut Board 341 Walnut Research Reports 2013

18 Figure 1. A map of the California State showing the distribution of the various species of Botryopshaeriaceae and Diaportheciae (species of Phomopsis ) isolated from walnut samples during the last few years. California Walnut Board 342 Walnut Research Reports 2013

19 Fontelis Luna Sensation Luna Exper Pristine Quilt Xcel Quadris Top Abound Control -1 Control -2 A Fontelis Luna Sensation Luna Exper Pristine Quilt Xcel Quadris Top Abound Control -1 Control -2 B Infected shoots (%) Infected peduncles (%) C 60 D Fontelis Luna Sensation Luna Exper Pristine Quilt Xcel Quadris Top Abound Botryosphaeria (%) Control - Average Infected husks (%) Shoots Peduncles Husks Figure 2. Efficacy of fungicides against Botryosphaeria canker and blight measured as incidence of Botryosphaeriaceae recovered from plated current-growth tissues (shoots, peduncles, and husks) on the surface of acidified PDA; samples were collected from a Butte Co. walnut orchard on October 25, California Walnut Board 343 Walnut Research Reports 2013

20 Quilt Xcel Luna Sensation Tilt Quadris Top Quash Pris/Luna Exp/Quadris Fontelis Pristine Control -1 Control -2 A Luna Exper Quilt Xcel Luna Sensation Tilt Quadris Top Quash Pris/Luna Exp/Quadris Fontelis Pristine Control -1 Control -2 B Infected shoots (%) Infected peduncles (%) Luna Exper Quilt Xcel Luna Sensation Tilt Quadris Top Quash Pris/Luna Exp/Quadris Fontelis Pristine Control -1 Control -2 C Botryosphaeria (%) D Infected husks (%) 0 Shoots Peduncles Husks Figure 3. Efficacy of fungicides against Botryosphaeria canker and blight measured as incidence of Botryosphaeriaceae recovered from plated current-growth tissues (shoots, peduncles, and husks) on the surface of acidified PDA; samples were collected from a Tehama Co. walnut orchard on October 25, California Walnut Board 344 Walnut Research Reports 2013

21 Figure 4. The life cycle of the anthracnose disease in walnuts was constructed based on observations of infections of leaves, petioles, shoots, and fruit of walnut and examination of overwintered leaves collected from the orchard floor where perithecia of Gnomonia leptostyla were found. Infections were also found on secondary growth of walnuts late in summer, suggesting secondary infection cycles (by T.J. Michailides & W. Coates). California Walnut Board 345 Walnut Research Reports 2013

22 APPENDIX I Protocol of BUD MONITORING (BUDMON) to detect and predict Botryosphaeria canker and blight in walnut (adapted from the pistachio BUDMON Technique) 1. Randomly collect 100 flower (male and female) walnut buds per orchard. 2. Place buds in a nylon mesh pouch. 2. Surface sterilize buds in 10% bleach solution [90 ml water + 10 ml bleach (5.25% sodium hypochlorite)] + 1 drop of a surfactant (or a drop of a dish soap). 3. Agitate the bag containing the buds in this solution for 3 minutes. 4. Dip the bag with buds in sterile water (optional) 5. Use new solution for 5-6 separate bud samples. 6. Allow buds to dry for about 10 minutes over clean paper towels in a positive flow hood. 7. Place buds on surface of soft acidified potato-dextrose agar (APDA) in Petri plates; 10 buds per plate. 8. Incubate plates at 25-27ºC (77 to 80ºF) for 5 to 7 days. 9. Mark plates on the under surface on buds showing growth of Botryosphaeriaceae species after 3 days. 10. Do a final count after 5 to 7 days incubation. 11. Calculate % buds infested/infected with Botryosphaeriaceae. California Walnut Board 346 Walnut Research Reports 2013