EPIDEMIOLOGY AND MANAGEMENT OF BOTRYOSPHAERIA / PHOMOPSIS CANKER AND BLIGHT AND ANTHRACNOSE BLIGHT OF WALNUT IN CALIFORNIA Themis Michailides , David P . Morgan , Daniel G . Felts , Yong Luo , Ryan Puckett , Janine Hasey , Bruce Lampinen , Emily Symmes , William Coates , Richard Buchner , Dani Lightle , Elizabeth Fichtner , David Rodriguez , and Connor Cunningham ABSTRACT The Botryosphaeriaceae fungi and Phomopsis species are now widely distributed in walnut orchards in all the counties where walnuts are grown . In fact , 20 % of the samples were diagnosed infected by Botryosphaeria , 20 % infected by Phomopsis , and the majority of the remaining samples had both Botryosphaeria and Phomopsis together while only a few of the samples were diagnosed as branch wilt caused by Neoscytalidium dimidiatum ( the former branch wilt fungus Hendersonula toruloidea ) . Furthermore , in some cases the branch wilt fungus killed entire 1 to 2 year old trees in the field . Again , in 2016 , for both Botryosphaeria and Phomopsis , we confirmed the presence of both reproductive structures , pycnidia producing the water - splashed conidia and perithecia producing the airborne ascospores . This information is of major importance because it helps us understand how these fungal pathogens spread within and among the walnut orchards and among other susceptible agricultural and riparian tree and bush species . Although conventional methods of trapping the spores of these fungal pathogens under drought conditions are not sensitive enough to detect low amounts of released spores , the developed effective and very sensitive quantitative PCR method ( qPCR ) can detect low amounts of spores trapped with spore traps after a rain event . Spores were detected for the major fungi causing Bot canker and blight in a walnut orchard from May through mid - September . An experiment simulating a rain of 8 hours showed that the majority of pycnidiospores of Neofusicoccum are released within the first 2 hours after the initiation of the rain and the pycnidia are exhausted of the majority of the spores within the first 4 hours of the rain . Pruning wounds of walnut remain susceptible to infection by the Botryosphaeriaceae fungi for at least 4 months after the wounds are made . Shoots of 3 - to 4 - years - old wood are more susceptible and developed larger cankers than those of 1 - to 2 - years - old shoots ( confirmed for a 2nd year ) . The majority of fungicides used on cuts immediately after pruning did not protect the wounds from infection . Only Merivon significantly reduced the percentage of cankers in shoots pruned and inoculated with Lasiodiplodia citricola in October 2015 ( fall pruning ) ; this effect was not seen in February 2015 prunings ( winter pruning ) or with Neofusicoccum parvum inoculations at both pruning dates . In fungicide spray experiments , all of the treatments with Merivon at various timings significantly reduced blighted fruit . The best Merivon ® treatment was four sprays at bloom ( 31 March ) , on 5 May , 10 June , and 12 July . Among a number of treatments with different timings with Merivon ® , the calendar - scheduled program ( conventional ) , the timing Experiment , and the spray program predicated by the Leaf Wetness Model ( LWM ) proved most effective . The LWM triggered 3 sprays each done before or immediately after an infection event ( at least 50 ° F and at least ¼ of an inch wetness ) . We also provide evidence again that the majority of the fungicide sprays applied in spring and summer have a long - term effect ( approximately 6 to 8 months after application ) on reducing Botryosphaeria canker and blight significantly as determined by the pre - season BUDMON assay . Three sprays in the spring ( 5 April to 1 June ) of a number of fungicides showed efficacy against California Walnut Board the anthracnose of walnut ( second year efficacy data against this disease ) . Interestingly and to our surprise , a few specific fungicides and combinations of fungicides with biologicals ( see Table 5 for details ) reduced significantly walnut blight similarly to the standard grower spray with Badge + Manzate . OBJECTIVES 1 . To continue surveying walnut orchards and diagnosing diseased samples brought / sent to the laboratory by growers , farm advisors , pest control advisers , and other industry representatives . 2 . To monitor the dynamics of inoculum over the seasons in walnut orchards . 3 . To investigate latent ( symptomless ) infection of buds , shoots and fruit as a tool to predict disease risk in orchards . 4 . To determine the relationship of pruning wounds to susceptibility to infection . 5 . To determine the efficacy and best timing of fungicides to control Botryosphaeria and anthracnose blights in walnut . SIGNIFICANT FINDINGS 1 . Confirm the presence of two types of inoculum ( water - splashed conidia and air - borne ascospores ) produced by Botryosphaeria and Phomopsis species in walnut orchard . 2 . The quantitative PCR method detected Phomopsis only in April , while Botryosphaeria dothidea , Lasiodiplodia , Cytospora and Neofusicoccum spp . from April to August / September ; and Diplodia spp . were found only sporadically . 3 . Higher incidence and greater severity of cankers developed on spurs after fruit inoculation with Lasiodiplodia citricola but not with Neofusicoccum parvum with late - season inoculations than early – season inoculations ( linear relationship ) . 4 . Newly - emerged ( spurs ) / shoots harbor latent infection by Phomopsis , Botryosphaeria dothidea , and species of Lasiodiplodia , Neofusicoccum , and Cytospora , but not Diplodia . The results were confirmed with the quantitative qPCR assay , 5 . The qPCR worked well and detected the latent infections on fruit . The molecular severity levels corresponded well with the incidence of latent infection on fruit , newly - developed spurs , and 1 - year - old shoots . 6 . Initially the 1 - year - old shoots had higher levels of latent infection , but this difference diminished as the season progressed . 7 . We confirmed that pruning wounds are susceptible for at least 4 months , and the wounds of 3 - and 4 - year - old shoots favor the development of larger cankers than the 1 - and 2 - year - old shoots . 8 . Treating pruning wounds created in February ( winter pruning ) and inoculating with Lasiodiplodia citricola 1 day after fungicide treatment was not effective in reducing either infection or canker length . 9 . For the fall ( 29 Oct ) pruning though , the fungicide Merivon protected the pruning wounds from infection . 10 . Several fungicide calendar sprays done on 6 May , 10 June , 12 July , and 2 August reduced significantly the incidence of infected fruit and cankered spurs ( see Table 1 for details ) . California Walnut Board 11 . We confirmed that a bloom spray ( 31 March ) reduces Botryosphaeria / Phomopsis canker and blight . 12 . Sprays in June , July , or August with Merivon provided numerically the best control . 13 . The leaf wetness model ( LWM ) predicated 3 sprays ( 26 April , 23 May , and 20 June ) provided similar control as the 4 calendar sprays . 14 . Several fungicides applied at the ½ leaf size ( April 5 ) to 1 June ( 3 total sprays ) provided a second year good efficacy results against anthracnose of walnut . 15 . Surprisingly , Luna Experience + Serenade Opti ( ! ) , the unregistered Kenja ( ! ) ( isofetamid ) fungicide , Luna Experience + Movento applied on 5 April , 28 April , and 1 June controlled walnut blight equally well as the standard treatment Badge + Manzate applied on the same dates . 16 . Other fungicide treatments were less effective but showed significantly lower walnut blight than the untreated control , suggesting that sprays done against Botryosphaeria canker and blight also help to reduce walnut blight . 17 . We confirmed that the majority of the fungicide treatments have a long term effect in reducing Botryosphaeria in walnut buds and some of them also reduce Phomopsis . PROCEDURES 1 ) To continue the survey of walnut orchards and diagnose diseased samples brought / sent to the laboratory by growers , farm advisors , and pest control advisers . We continued to diagnose samples brought to the laboratory by walnut growers , farm advisors , pest control advisers , and representatives of various chemical companies . We like this activity because we feel we provide walnut growers with direct and timely help , and advice on the diagnosis of disease in their walnut orchards , giving them the opportunity to design and / or adjust pest management approaches accordingly . Diagnosis is done “ on the spot ” , either by direct observation , examining spurs for the presence of pycnidia , by plating the margins of cankered tissues , or by plating samples of random buds in early spring by using the BUDMON Technique . Diagnostic results are communicated back to the interested parties either by a phone call or mainly by an e - mail message so that the grower has also a record of the diagnostic results . 2 ) To monitor the dynamics of inoculum over the seasons in walnut orchards . In this year , we continuously focused on identification of canker - causing pathogens and their corresponding quantities as inoculum in the air . We quantified the dynamics of the inoculum in a Chandler walnut orchard with history of severe disease found immediately next to the Sacramento River . We developed molecular approaches by using real - time ( quantitative ) PCR ( qPCR ) to by using SYBR method to identify and distinguish six canker - causing fungal species , including Phomopsis spp . , Botryosphaeria dothidea , Lasiodiplodia spp , Cytospora spp . , Neofusicoccum spp . , and Diplodia spp ( Luo et al , 2017 ) . The standard curves from this study were used to quantify the inoculum in the air and to quantify latent infection levels of walnut shoots as follows . Two Burkard spore traps ( Cyclone sampler for field operation ( Burkard Manufacturing Co . Ltd , Rickmansworth , Hertfordshire , UK ) were placed in two different areas ( namely East and West ) in a walnut orchard located next to Sacramento River in Butte County of California . The air samples were collected approximately every 3 to 4 weeks into a 1.6 ml centrifuge tube . The velocity of the California Walnut Board air during operation of the trap was approximately 11520 L of air / day . One ml of distilled water was added in each sample tube and processed as described below . The spore trap samples were processed in 2016 by using the sensitive qPCR method developed in this study . For each spore trap sample , 200 µl of each of the original spore suspension was used to extract DNA , and the qPCR systems were applied to quantify the number of spores as log 10 ( number of spores / day ) for each pathogen . 3 ) To investigate latent ( symptomless ) infection of buds , shoots and fruit as a tool to predict disease risk in orchards . Latent infection of walnut fruit . To determine when hulls are infected , first immature and then maturing Vina fruit were inoculated in the field with Lasiodiplodia citricola ( isol . 6 - I35 ) and Neofusicoccum parvum ( isol . 1 - L87 ) , using 7 fruit per isolate on each of 3 trees . Inoculations were done every 3 weeks , beginning May 12 , 2015 with the last inoculation done Sept 15 , 2015 . Shoots with fruit were sprayed with a 15,000 - 50,000 spores / ml suspension of Lasiodiplodia citricola or Neofusicoccum parvum . The inoculations were performed in late afternoon , bagged with a plastic bag to maintain humidity , and covered in a white paper bag to prevent sunburn and overheating . Bags were removed early the following morning . Inoculated fruit from the first five inoculation dates were collected Sept 24 , 2015 ; fruit from the last two inoculations were collected on Oct 26 , 2015 . Blighted fruit were collected and recorded for each treatment and isolations made to determine if the pathogen used for inoculation could be recovered . The results from the fruit were presented in last year’s report . We collected these shoots March 22 , 2016 and measured cankers that had developed after inoculation . Similar inoculations were made May through September of 2016 . Pathogens used this year were Lasiodiplodia citricola , Neofusicoccum parvum , and Phomopsis sp . The number of inoculations was increased , with 10 fruit clusters on each of three trees for each pathogen . Fruit were removed from the clusters in early November but isolations have not been done . The shoots will be collected in March for canker measurement . Quantification of latent infection level of shoots and fruit using molecular approach . In the same orchard used in Objective 2 , shoots were collected periodically in April , July and October of 2016 . For each sampling , about 10 - 20 cm - long shoots , including the newly - emerged shoots and the top portion of the old shoot in the proximity of the new shoot , ( usually 1 - year old ) were collected . Thus , each sample contained two parts : new shoots and old shoots , and processed separately . For each sampling , 32 such shoots were randomly collected . These shoots were washed twice with tap water , soaked in 10 % commercial bleach for 10 min for surface sterilization , washed three times again , and air dried for 2 days . A pencil sharpener was used to grind shoot samples into fine wood pieces which were used to extract DNA by using the FastDNA kit ( MP Biomedical , CA ) . Briefly , the pathogen group - specific primers were used in real - time PCR to obtain the corresponding Ct values . The published equation of standard curve for each pathogen group ( Luo et al . , 2017 ) was used to calculate the DNA quantity for each pathogen in each sample . To quantify the infection level of shoots , we introduced the concept of molecular severity ( MS ) : MS = Log10 ( P / H ) , where P is the weight of the pathogen’s DNA in femtograms ( fg ) , which is California Walnut Board calculated by using the equation of the standard curve for the corresponding pathogen ( Luo et al . , 2017 ) based on the Ct value from its reaction with the corresponding primers , and where H is the shoot weight in grams ( g ) . Thus , if the minimum detectable amount of pathogen DNA in one gram of shoot is theoretically assigned as 1 fg , the MS would be 0 . The maximum of the amount of pathogen DNA in one gram of shoot tissue could be theoretically one gram ( = 1015 fg ) , and the maximum value of MS should be 15 . Thus , the range of MS value is 0 - 15 . However , since when no infection is detected we assign MS = 0 , the theoretically detectable amount of pathogen DNA in one gram of shoot should be > 1fg . The concept and calculation of MS were used to determine the infection level for all the shoot samples used in this study . The incidence of latent infection in terms of proportion of shoots showing positive results over all detected shoots was obtained for each pathogen on each sampling date . Average MS from 32 shoot samples was obtained for each pathogen and each sampling date . Comparisons in MS between new and old shoots were conducted for each pathogen and on each sampling date . Similarly , latent infection levels in fruit were determined in May of 2016 for the orchard in Butte Co . mentioned above and the walnut orchard in KARE . In early May of 2016 , 30 fruit and 24 fruit were randomly collected from the walnut orchards in Butte Co . and KARE , respectively . The same approaches using qPCR assay mentioned above were applied . The incidences of latent infection and molecular severities ( MS ) for both orchards were quantified . 4 ) To determine susceptibility of pruning wounds to infection . To determine whether and how long pruning wounds are susceptible to infection , one of the most aggressive ( virulent ) species , Lasiodiplodia citricola ( isol . 6 - I35 ) was used for this experiment . The Chandler walnut cultivar at Kearney experimental orchard was pruned as follows : Groups of 10 each from 1 - , 2 - , 3 - , and 4 - year - old shoots were pruned and inoculated . Inoculations with a spore suspension of 50,000 spores / ml were done with post - pruning intervals of 0 days , 3 days , and 1 , 2 , 3 , 4 , 8 , 12 , and 16 weeks . This experiment began on 9 February 2015 . Ten non - inoculated pruned shoots from each age category served as the control treatment . Shoots were collected and cankers measured March 7 - 10 , 2016 . ( Note : 10 shoots × 9 inoculation dates × 4 age categories + 40 control shoots = a total of 400 shoots were used in this experiment . ) The pruning and inoculation procedure was carried out twice in 2015 . The second experiment began on 27 October 2015 and the inoculation schedule was as described for the February pruning . Cankers were measured after collecting the shoots . Isolations were taken from fall pruning cankers and the cankers were only counted if the inoculated pathogen was recovered . Isolations were not taken from the spring pruning . Canker lengths were recorded but results are presented in relation to the percentage of inoculations that resulted in canker formation . 5 ) To manage Botryosphaeria canker and blight and anthracnose blight in walnut ( fungicide trials ) . Protecting pruning wounds : Potential fungicides used to protect pruning wounds of walnuts were tested for their efficacy against infection by Botryosphaeriaceae . Chandler trees in our experimental orchard at Kearney were used in this experiment . Shoots of age 1 - , 2 - , 3 - , and 4 - years were pruned as described in objective # 4 above . Immediately after pruning , wounds were treated with one of the following fungicides : Merivon , Merivon + VitiSeal , Quadris Top , or Abound . Five shoots of each age were used per treatment , for a total of 200 shoots pruned on Feb 12 , 2015 , then treated with fungicide , except for 10 shoots in each age category which served as controls . The day following the treatments , 5 shoots in each group were inoculated with Lasiodiplodia and 5 with Neofusicoccum , including the untreated control shoots . Cankers were measured on 7 March 2016 . The Merivon and the control treatments as described above were repeated to compare spring and fall pruning times . Pruning was done on 29 Oct and inoculation on 30 Oct 2015 . Cankers were measured on 17 November 2016 . Canker lengths were recorded but results are presented in relation to the percentage of inoculations that resulted in canker formation . Fungicide concentrations were based on applying the field rate of oz / acre in 5 gallons of water per acre . These rates were as follows : Merivon = 6.8 oz / acre ; Quadris Top = 14 oz / acre ; Abound = 12 oz / acre ; Viti Seal 1 : 10 dilution of the commercial product . Fungicide trial in Butte County . We established a fungicide trial in Butte Co . in 2016 . A Chandler orchard located adjacent to the Sacramento River which had a history of severe infection in 2014 was used . The following experiments were done in this orchard : a ) a trial for the efficacy of various fungicide treatments based on a calendar spray ; b ) a trial with sprays based on the Bot infection model ( Morgan et al . , 2010 ) ; c ) a trial to determine the “ best timing ” by spraying Merivon only once at different dates to different sets of 5 trees ; and d ) a trial to compare efficacy of Merivon bloom spray only with the Merivon postharvest spray only to determine whether such sprays ( bloom or postharvest ) have any efficacy against reducing the disease . All the trees used for the above fungicide trials were randomized in this orchard using 5 replications in 5 different rows of trees . All the fungicides were sprayed with a handgun using our 4 - tank sprayer . Fruit with black hulls were counted on the ground after commercial shaking , on 28 September 2016 , the day of harvest . The incidence of blighted current season shoots was recorded using pruning towers on 5 and 6 December 2016 . Furthermore , 25 buds per tree ( a total of 125 buds per treatment ) will be collected in Feb / March 2016 to perform the BUDMON assay and determine the long effect of fungicides on infestation of buds by species of Botryosphaeriaceae and / or infection by Phomopsis . Fungicide trial in Yuba County . This trial was set up in a Vina orchard in cooperation with cooperative extension farm advisor Janine Hasey . Merivon was sprayed at 6.5 fl oz / A with 3 fl oz / A organic silicone at 100 gallons / acre at various timings . There were four replicates consisting of entire tree rows with buffer rows between treated rows . All the fungicides sprays were done using the grower’s commercial rig . Buds for the BUDMON assay were collected in February 2016 to determine the effects of fungicides ( Fig . 21 - 22 ) . Fungicide trial in Colusa County . This trial was set up in a Chandler orchard in cooperation with farm advisor Janine Hasey . The fungicides were sprayed using the grower’s commercial rig . There were four replicates consisting of entire tree rows with buffer rows between treated rows . Buds for the BUDMON assay were collected in February 2016 to determine the effects of fungicides ( Fig . 23 - 24 ) . Fungicide trial in Colusa County — Nickels Soil Lab . This trial was set up in a Chandler orchard at Nickels Soil Lab in cooperation with farm advisor Janine Hasey and specialist Bruce Lampinen . The fungicides were hand sprayed to run - off on pruning wounds using spray bottles . Primary comparisons in this experiment are a hedging trial vs . a whisking ( lateral limbs removed on north and south sides , ~ 10 cuts / tree ) trial , both pruned on 28 March 2016 . In the hedging trial there are five prunings per treatment in each of six reps . In the whisking trial there are three prunings per treatment in each of four reps . The treatments are : 1 ) untreated control ; 2 ) Quadris Top , 14 fl oz / ac ; 3 ) ; Quadris Top + Viti - Seal , 14 fl oz / ac ; 1 : 50 ; 4 ) Quadris Top + Spur Shield , 14 fl oz / ac ; 1 : 50 ; 5 ) Tilt , 8 fl oz / ac ; 6 ) Tilt + Viti - Seal , 8 fl oz / ac ; 1 : 50 ; 7 ) Tilt + Spur Shield , 8 fl oz / ac ; 1 : 50 . One year after treatment , cankers will be measured on treatment and control limbs and samples taken to identify Botryosphaeria and Phomopsis species present on dead limbs in the orchard . Fungicide trial against anthracnose in San Benito County . The walnut anthracnose disease , caused by the fungus Gnomonia leptostyla , the perfect stage of the fungus Marssonina juglandis , has created some problems in walnut cultivars in commercial orchards and walnut rootstock ( i.e . , black walnut ) in nurseries in the past under normal , yearly rainfall . Because the anthracnose disease was very severe in walnut orchards in San Benito County in 2011 and 2012 , we easily found both the conidia and the ascosporic stages of the fungus in an orchard which the grower allowed us to use for a fungicide trial . The conidia are produced in saucer - like structures produced on leaves , hulls of fruit , petioles , and shoots . The perithecia ( which produce the airborne ascospores of the fungus ) were found in leaves on the ground in the spring . When the perithecia become wet from sprinkler irrigation or rain , the ascospores are shot into the air and become airborne . In 2016 , we established a fungicide efficacy trial . The fungicides used , the label rates , and dates of sprays are listed in Table 5 . Because of the many treatments we had to use in 2016 , we reduced the number of 4 replicated trees to 3 trees per fungicide treatment . Residual activity in 2016 of fungicides sprayed in the spring of 2015 . The long term activity of fungicides against Botryosphaeriaceae species was studied because previous research showed that fungicide sprays resulted in reduction of primary inoculum of apple powdery mildew during the season following the application season . We evaluated the level of bud infection / infestation by Botryosphaeria and / or Phomopsis during the late winter of 2016 about 8 months from the time the last spray was applied on to the trees in 2015 . Fifty dormant buds were collected on five 2015 fungicide trials in walnut orchards located in Butte ( one south of Chico , and another east of Durham ) a third in Colusa , a fourth in Yuba , and a fifth in San Benito counties . Buds were plated on acidified PDA according to the BUDMON technique and plates incubated at 27ºC ( 81ºF ) . The incidences of Botryosphaeria / Phomopsis were recorded 7 days after plating and incubating the plates . RESULTS AND DISCUSSION 1 ) To continue the diagnosis of diseased samples brought or sent to the laboratory by growers , farm advisors , pest control advisers , and industry representatives . We diagnosed 34 walnut samples in 2016 . Of these , 7 samples produced species of Botryosphaeriaceae and 7 were found to have Phomopsis species ; some of these samples had both pathogens . The majority of these samples had pycnidia of these fungi , fruiting structures produced in infected plant tissues underneath the epidermal layer producing pycnidiospores . Pycnidiospores require water to exude from the pycnidia and spread to infect plant tissues . In four of these 2015 samples that involved blighted shoots at least 1 year old ( 3 from Butte Co and one from Tulare Co . ) , in addition to the pycnidia of Botryosphaeria and Phomopsis , we also found 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 . California Walnut Board 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 and in shoots collected from trees . The perfect state of Phomopsis species belongs in the genus Diaporthe . At least 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 . This is important information because it helps to realize how these fungal pathogens spread among walnut orchards . The diagnosis of each sample was communicated on time to farm advisors , pest control advisers , or growers who submitted samples in 2016 . 2 ) To monitor the dynamics of inoculum during the season in walnut orchards . The six pathogen groups were all found during the season , while Diplodia spp . was detected sporadically at very low level ( Figure 1 ) . The species of Phomopsis was detected only in the early stage in the orchard and not found later in the season ( Fig . 1 ) . In the air , B , dothidea , Lasiodiplodia app . Cytospora spp . and Neofusicoccum spp . were the dominant pathogens in the orchard . These four pathogen groups were detected in most sampling periods , indicating the major component of canker - causing pathogens in walnut orchard . Generally , the quantities of B . dothidea were higher than those of other pathogens in different sampling periods . The spore densities of these pathogens ranged from several tens to several hundreds of spores per day ( log10 ( no . spores / day ) = 1 to 2.5 ) . Although Cytospora is not reported as a pathogen of walnut in California , there were a lot of spores of this pathogens collected by the traps in the orchard . This is most likely spore inoculum produced in susceptible hosts , such as prunes , almonds , and riparian tree species that are abound in the vicinity where the walnut orchard is located . California Walnut Board 0.00 0.50 1.00 1.50 2.00 2.50 3.00 4 / 7 - 4 / 26 4 / 26 - 6 / 9 6 / 9 - 7 / 12 7 / 12 - 8 / 2 8 / 2 - 9 / 29 Phomopsis spp . 0.00 0.50 1.00 1.50 2.00 2.50 3.00 4 / 7 - 4 / 26 4 / 26 - 6 / 9 6 / 9 - 7 / 12 7 / 12 - 8 / 2 8 / 2 - 9 / 29 Botryosphaeria dothidea 0.00 0.50 1.00 1.50 2.00 2.50 3.00 4 / 7 - 4 / 26 4 / 26 - 6 / 9 6 / 9 - 7 / 12 7 / 12 - 8 / 2 8 / 2 - 9 / 29 Lasiodiplodia spp . 0.00 0.50 1.00 1.50 2.00 2.50 3.00 4 / 7 - 4 / 26 4 / 26 - 6 / 9 6 / 9 - 7 / 12 7 / 12 - 8 / 2 8 / 2 - 9 / 29 Cytospora spp . 0.00 0.50 1.00 1.50 2.00 2.50 3.00 4 / 7 - 4 / 26 4 / 26 - 6 / 9 6 / 9 - 7 / 12 7 / 12 - 8 / 2 8 / 2 - 9 / 29 East West Neofusicoccum spp . 0.00 0.50 1.00 1.50 2.00 2.50 3.00 4 / 7 - 4 / 26 4 / 26 - 6 / 9 6 / 9 - 7 / 12 7 / 12 - 8 / 2 8 / 2 - 9 / 29 East West Diplodia spp . Period of time in 2016 Log 10 ( no . of spores / day ) Figure 1 . Spore densities of six canker - causing pathogens in air samples collected with two Burkhart spore traps ( East trap and West trap ) installed in a Chandler walnut orchard in Butte Co . Spore densities were quantified with the qPCR method developed in this study . Five samplings were conducted , each representing and containing the total spores in the air collected during a period of time in 2016 . The specific primer pairs were used to quantify the density of spores of the corresponding species in the same samples , in terms of total number of spores collected from spore trap per day in log values . 3 ) To investigate latent ( symptomless ) infection of buds and shoots as a tool to predict disease risk in orchards . Latent infection of walnut fruit . Data reported last year showed that fruit inoculations from May to September resulted in a continuous infection and blighted fruit , suggesting that under favorable conditions immature fruit can be infected latently and fruit will blight by harvest time . Inoculations late in the season resulted in significantly higher fruit blight than the early inoculations did ( data not shown ) . Shoots from fruit inoculations were evaluated for cankers . Percentage of cankers developed from the Lasiodiplodia citricola inoculations showed an increase over the course of the season ( Fig . 2 ) with a strong correlation to the time of inoculation . A similar trend was found for canker length ( Fig . 3 ) as well , though the correlation was not as good . Cankers from Neofusicoccum parvum inoculations were not found to be correlated with inoculation date . Figure 2 . Percentage of Vina shoots with cankers from inoculations of fruit clusters with Lasiodiplodia citricola or Neofusicoccum parvum at 21 day intervals , beginning May 12 , 2015 and ending Sep 15 , 2015 . Inoculations dates are shown as Julian dates ; the cankers were recorded Mar 22 , 2016 . Figure 3 . Average length of cankers from Vina shoots with fruit clusters inoculated with Lasiodiplodia citricola or Neofusicoccum parvum at 21 day intervals , beginning May 12 , 2015 and ending Sep 15 , 2015 . Inoculation dates are shown as Julian dates ; the cankers were recorded Mar 22 , 2016 . Quantification of latent infection level of shoots using molecular approach . Diplodia species were not detected in the walnut shoots in any of the seasonal collected samples ( Fig . 4 ) . The incidence of species of Phomopsis in newly - emerged shoots was much lower than those of old shoots ( one - year old ) ( Fig . 1 ) , indicating very low frequency of latent infection by this pathogen in new shoots compared with the old shoots . Shoots with latent infections by B . dothidea , Lasiodiplodia spp . Cytospora spp . and Neofusicoccum spp . were all detected with various incidences ( Fig . 4 ) . 0.00 0.20 0.40 0.60 0.80 1.00 April July October Phomopsis spp . 0.00 0.20 0.40 0.60 0.80 1.00 April July October Botryosphaeria dothidea 0.00 0.20 0.40 0.60 0.80 1.00 April July October 0.00 0.20 0.40 0.60 0.80 1.00 April July October Cytospora spp . 0.00 0.20 0.40 0.60 0.80 1.00 April July October New shoot One - year shoot Neofusicoccum spp . 0.00 0.20 0.40 0.60 0.80 1.00 April July October New shoot One - year shoot Diplodia spp . Lasiodiplodia spp . Incidence of lantent infection of shoots Month of 2016 However in comparison , the incidences of latent infection of shoots by Cytospora spp . seemed lower than those of other three species in July and October ( Fig . 4 ) . Figure 4 . Incidences of latent infections of newly - emerged shoots and old , 1 - year shoots caused by six canker - pathogen groups in samples collected in April , July , and October from a commercial walnut orchard in Butte County . The real - time PCR assay was applied to process these shoot samples . Comparison in Molecular Severity ( MS ) between new and old shoots demonstrated that the MSs were significant higher in old shoots than in new shoots in the samples collected in April for all the pathogens ( Fig . 5 ) . While there was no significant difference in MS between the new and old shoots for most samples in the later ( July and October ) samplings ( Fig . 5 ) . This trend was still maintained in the second ( in July ) sampling for Cytospora spp . and Neofusicoccum spp . , while variations in comparison in MS between newly - emerged and old shoots were found for the other pathogen groups and in other sampling periods ( Fig . 5 ) . The results indicated the possibility of pathogen movement from old to new shoots during the growing season and some endophytic features of pathogen species in walnut shoots that need further investigation . 0 2 4 6 8 10 April July October Phomopsis spp . a a b a a b 0 2 4 6 8 10 April July October Botryosphaeria dothidea a a b a a b 0 2 4 6 8 10 April July October Lasiodiplodia spp . a a a b a a 0 2 4 6 8 10 April July October Cytospora spp . a b a b a b 0 2 4 6 8 10 April July October New shoot One - year shoot Neofusicoccum spp . a a a b a b 0 2 4 6 8 10 April July October New shoot One - year shoot Diplodia spp . Month of 2016 Molecular severity of lantent infection of shoots The results from latent infection of shoots were quite consistent to those of inoculum quantification in the air in Objective 2 , in terms of component and dominance of the pathogen population in walnut orchard . Figure 5 . Comparison of mean molecular severities ( MS ) between newly - emerged shoots and 1 - year - old walnut shoots quantified for each of the six canker - causing pathogens . Three samplings were conducted in a commercial walnut orchard in Butte County . The real - time PCR was applied to obtain MS data and 32 shoots were processed for each sampling . Quantification of latent infection level of fruit using the molecular approach . Again , Diplodia spp . were not detected in fruit in either walnut orchards in Butte Co . or the one at Kearney ( KARE ) ( Fig . 6 ) . The incidence of fruit with latent infection by Phomopsis was lower than those of other four pathogens in the Butte Co . orchard ( Fig . 6A ) . In this orchard , the incidence of fruit with latent infection by B . dothidea was the highest , while those with Lasiodiplodia spp . Cytospora spp . and Neofusicoccum spp . were quite close ( Fig . 6A ) . The MS values of all the five pathogen groups were very close to each other with similar standard errors ( Fig . 6A ) . In the walnut orchard at Kearney , no infections by Phomopsis spp . were detected in fruit ( Fig . 6B ) . The incidences of the other four canker pathogen groups were quite similar ( Fig . 6B ) , indicating that B . dothidea , Lasiodiplodia spp . Cytospora spp . and Neofusicoccum were all pathogens which could cause infections of fruit early in the season . However , no disease by Cytospora on walnut fruit has been reported . 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 0.00 0.10 0.20 0.30 0.40 0.50 0.60 Phomopsis Botryospaeria dothides Lasiodiplodia spp . Cytospora Neofusicoccom spp . Diplodia Incidence MS Buttte Co . A 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 0.00 0.10 0.20 0.30 0.40 0.50 0.60 Phomopsis Botryospaeria dothides Lasiodiplodia spp . Cytospora Neofusicoccom spp . Diplodia Incidence MS KARE B Incidence of latent infection of fruit Molecular severity ( MS ) of fruit latent infection Canker - causing pathogen spp . spp . spp . spp . spp . spp . spp . spp . spp . spp . Figure 6 . Incidences of latent infection of walnut fruit and the corresponding molecular severities caused by six canker - causing pathogen groups for two walnut orchards quantified in May 2016 . A total of 30 and 24 fruit were randomly collected from walnut orchards in Butte Co . and KARE , respectively . The qPCR assay was applied in the quantification approach . 4 ) To determine the susceptibility of pruning wounds to infection by canker fungi . Figures 7a and 7b summarize the results of periodic Lasiodiplodia citricola inoculations of different age ( 1 - to 4 - years - old ) Chandler shoots , pruned in February and October respectively . Cankers were infected even sixteen weeks after pruning , which is in contrast to other tree species ( i.e . Prunus spp . ) whose pruning wounds usually remain susceptible to canker fungi for only two weeks after pruning . Overall , there were higher percentages of cankers for the February pruning dates than the fall dates . Figures 8a and 8b present the percentage cankers for each age of shoot in relation to inoculation date . The first inoculation resulted in a very high percentage of cankers in all age shoots in both the February and October prunings . The 3 - and the 4 - year - old shoots showed high numbers of cankers throughout the season for the February prunings but were more variable with significant declines in the October pruned group at the later inoculations . After pooling all the data of inoculation dates by age , it is apparent that the 3 - and 4 - year - old shoots showed significantly higher percentages of developed cankers than the 1 - and 2 - year - old shoots ( Figs . 9a and 9b ) . This was seen with both February and October prunings . Canker length data ( not shown ) exhibited a similar pattern as well . The long period of susceptibility of pruning wounds of walnut could be partially explained by the fact that there is a central channel in each shoot filled with loose pith tissues . Apparently , when spraying spore inoculum on the pruning wounds , fungal inoculum lands in this channel , is absorbed by the pith tissues , and therefore may survive long in the pith channel , initiating infection internally . Moreover , in the field , one could easily see that the Botryosphaeriaceae fungi move in advance of the canker ( killed tissues ) margins growing in the pith ; in some cases , this advanced growth in the pith tissues is more than 1 inch in length . Thus , to be safe , we suggest that when a grower tries to remove a cankered shoot by pruning , the cut should be done at least 2 inches below the characteristic margin of canker externally . Similar recommendations are given when growers prune cankered shoots of pistachio to manage the Botryosphaeria panicle and shoot blight disease . California Walnut Board 0 20 40 60 80 100 0 day a a a b 0 20 40 60 80 100 3 days a ab b b 0 20 40 60 80 100 1 week a ab b b 0 20 40 60 80 100 Control a ab ab b 0 20 40 60 80 100 2 weeks a a a a 0 20 40 60 80 100 3 weeks a ab b ab 0 20 40 60 80 100 4 weeks a a a a 0 20 40 60 80 100 8 weeks a a a a 0 20 40 60 80 100 1 year 2 years 3 years 4 years 12 weeks a a b b 0 20 40 60 80 100 1 year 2 years 3 years 4 years 16 weeks a a a a Age of pruned shoots Cankers in inoculated shoots ( % ) Figure 7a . Winter pruning and infection : Percentage of shoots with cankers in 1 - to 4 - years - old shoots inoculated periodically with Lasiodiplodia citricola after pruning . Shoots of cultivar Chandler were pruned on 9 - 10 Feb 2015 and inoculated from 0 days to 16 weeks after pruning , respectively . Cankers were evaluated during 4 - 7 Mar 2016 . ( There were 10 shoots per age per date of inoculation . ) 0.0 20.0 40.0 60.0 80.0 100.0 0 a a a a 0.0 20.0 40.0 60.0 80.0 100.0 3 days ab bc c 0.0 20.0 40.0 60.0 80.0 100.0 1 week ab a b b 0.0 20.0 40.0 60.0 80.0 100.0 Control ab a bc c 0.0 20.0 40.0 60.0 80.0 100.0 2 weeks a a ab b 0.0 20.0 40.0 60.0 80.0 100.0 3 weeks a a b b 0.0 20.0 40.0 60.0 80.0 100.0 4 weeks a a a a 0.0 20.0 40.0 60.0 80.0 100.0 8 weeks a a a a 0.0 20.0 40.0 60.0 80.0 100.0 1 year 2 years 3 years 4 years 12 weeks a a a b 0.0 20.0 40.0 60.0 80.0 100.0 1 year 2 years 3 years 4 years 16 weeks a a b a Age of pruned shoots Cankers in inoculated shoots ( % ) Figure 7b . Fall pruning and infection : Percentage of shoots with cankers in 1 - to 4 - years - old shoots inoculated periodically with Lasiodiplodia citricola after pruning . Shoots of the cultivar Chandler were pruned on 27 - 28 Oct 2015 and inoculated from 0 days to 16 weeks after pruning , respectively . Cankers were evaluated on 14 Nov 14 2016 . 0 20 40 60 80 100 0 20 40 60 80 100 120 1 year a bc bc c bc bc ab bc bc 0 20 40 60 80 100 0 20 40 60 80 100 120 2 years b ab ab ab ab a b ab 0 20 40 60 80 100 0 20 40 60 80 100 120 3 years b b b b b b b b a 0 20 40 60 80 100 0 20 40 60 80 100 120 4 years a a a a a a a a a Cankers in inoculated shoots ( % ) Inoculation - - days after pruning b 0 20 40 60 80 100 0 20 40 60 80 100 120 1 year a a a a a a b a 0 20 40 60 80 100 0 20 40 60 80 100 120 2 years a c ab a a b b ab b 0 20 40 60 80 100 0 20 40 60 80 100 120 3 years a - c c a - c bc ab a - c bc a a - c 0 20 40 60 80 100 0 20 40 60 80 100 120 4 years ab a ab b ab b a b b Cankers in inoculated shoots ( % ) Inoculation - - days after pruning a Figure 8a . Winter pruning and infection : Percentage of Chandler walnut shoots that developed cankers after inoculation with Lasiodiplodia citricola from 0 to 112 days after pruning . Shoots of ages 1 - to 4 - years - old were used in these inoculations ; pruning was done on 9 - 10 Feb 2015 ; evaluation done during 4 - 7 Mar 2016 . Figure 8b . Fall pruning and infection : Percentage of Chandler walnut shoots that developed cankers after inoculation with Lasiodiplodia citricola from 0 to 112 days after pruning . Shoots of ages 1 - to 4 - years - old were used in these inoculations ; pruning was done during 27 - 28 Oct 2015 ; and evaluation was done on 14 Nov 2016 . Figure 9a . Winter pruning and infection : Percentage of pruned 1 - to 4 - years - old Chandler walnut shoots that developed cankers after inoculation with Lasiodiplodia citricola spores . The average value for each year was from 10 inoculation dates after the 9 - 10 Feb 2015 pruning ; measurements were taken during 4 - 7 Mar 2016 . Figure 9b . Fall pruning and infection : Percentage of pruned 1 - to 4 - years - old Chandler walnut shoots that developed cankers after inoculation with Lasiodiplodia citricola spores . The average value for each year was from 10 inoculation dates after the 27 - 28 Oct 2015 pruning ; measurements were taken on 14 Nov 14 2016 . 5 ) To determine efficacy and best timing of fungicide sprays to control Botryosphaeria and anthracnose blights in walnut . Protecting pruning wounds . In the February , 2015 in the pruning experiment with Lasiodiplodia citricola and Neofusicoccum parvum inoculations , there was no significant difference in canker development among shoots of the different ages or for any of the fungicide treatments ( Figure 10a , 10b , 11a , 11b ) . In addition , there was no significant difference in canker length among the fungicide treatments . The results in 2016 showed no effect of the fungicides used in this experiment in reducing canker development as compared to the inoculated but untreated pruning wounds . ControlQuadris TopMerivonMerivon + VitiSealAbound Cankers in inoculated shoots ( % ) 0102030405060708090100 1 year 2 year 3 year 4 year aaaaaaaaaaaaaaabaaaaaa Figure 10a . Winter pruning and infection : Comparison within treatments of percentage of cankers from pruned shoots of 4 different ages ( 1 - to 4 - years - old ) inoculated with Lasiodiplodia citricola . Shoots were pruned and treated with fungicide 12 Feb 2015 and inoculated the following day ( 13 Feb ) ; cankers were measured on 7 Mar 2016 . California Walnut Board 0 20 40 60 80 100 Control Abound Merivon Merivon + VitiSeal Quadris Top 1 year a a a a a 0 20 40 60 80 100 Control Abound Merivon Merivon + VitiSeal Quadris Top 2 years a a a a a 0 20 40 60 80 100 Control Abound Merivon Merivon + VitiSeal Quadris Top 3 years a a a a a 0 20 40 60 80 100 Control Abound Merivon Merivon + VitiSeal Quadris Top 4 years a a a a a Fungicide treatment Cankers in inoculated shoots ( % ) Figure 10b . Winter pruning and infection : Comparison of percentage of cankers within each of the 4 different shoot - age categories ( 1 - to 4 - years - old ) inoculated with Lasiodiplodia citricola for each fungicide treatment . Shoots were pruned and treated with fungicide on 12 Feb 2015 and inoculated the following day ; cankers were measured on 7 Mar 2016 . ControlQuadris TopMerivonMerivon + VitiSealAbound Cankers in inoculated shoots ( % ) 0102030405060708090100 1 year 2 year 3 year 4 year aaaaaaaaaaaaaaabaaaaaa Figure 11a . Winter pruning and infection : Comparison within treatments of percentage of cankers from pruned shoots of 4 different shoot - ages categories ( 1 - to 4 - years - old ) inoculated with Neofusicoccum parvum . Shoots were pruned and treated with fungicide on 12 Feb 2015 and inoculated the following day ; cankers were measured on 7 Mar 2016 . 0 20 40 60 80 100 Control Abound Merivon Merivon + VitiSeal Quadris Top 1 year a a a a a 0 20 40 60 80 100 Control Abound Merivon Merivon + VitiSeal Quadris Top 2 years a a a a a 0 20 40 60 80 100 Control Abound Merivon Merivon + VitiSeal Quadris Top 3 years a a a a a 0 20 40 60 80 100 Control Abound Merivon Merivon + VitiSeal Quadris Top 4 years a a a a a Fungicide treatment Cankers in inoculated shoots ( % ) Figure 11b . Winter pruning and infection : Comparison of percentage of cankers within each of 4 different shoot - age categories ( 1 - to 4 - years - old ) inoculated with Neofusicoccum parvum for each fungicide treatment . Shoots were pruned and treated with fungicide on 12 Feb 2015 and inoculated the following day ; cankers were measured on 7 Mar 7 2016 . In the October , 2015 pruning and fungicide experiment with Lasiodiplodia citricola and Neofusicoccum parvum inoculations , differences were found with some treatments . Only Merivon was used to treat inoculations . No significant difference in canker development between shoots of different ages for either control or Merivon treatments was found in the Lasiodiplodia citricola inoculations ( Figure 12a ) . No differences were seen between control and Merivon in the 1 year shoots and in the 2 year shoots . However , there was a significantly higher percentage of cankers in control and Merivon in both the 3 and 4 years shoots ( Fig . 12b ) . The Neofusicoccum parvum inoculations resulted in differences between the age categories in the control group but not in the Merivon treated shoots ( Fig . 13a ) . No differences were seen between control and Merivon in within any of the age categories ( Fig . 13b ) . These results only showed an effect of the Merivon reducing canker development in inoculations with Lasiodiplodia citricola in October and not in any of the Neofusicoccum parvum inoculations . Walnut scale is present at a high level in much of this orchard now and might have interacted or interfered with the effect of pruning and infection rates for both pathogens . ControlMerivon Cankers in inoculated shoots ( % ) 0102030405060708090100 1 year 2 year 3 year 4 year aaaaaaaa Figure 12a . Fall pruning and inoculation : Comparison within treatments of percentage of cankers from pruned shoots of 4 different ages ( 1 - to 4 - years - old ) inoculated with Lasiodiplodia citricola . Shoots were pruned and treated with fungicide on 29 Oct 2015 and inoculated the following day ; cankers were measured on 17 Nov 17 2016 . Figure 12b . Fall pruning and inoculation : Comparison of percentage of cankers within each of 4 different shoot age categories ( 1 - to 4 - years - old ) inoculated with Lasiodiplodia citricola for treatment and control . Shoots were pruned and treated with fungicide on 29 Oct and inoculated the following day ; cankers were measured on 17 Nov 2016 . ControlMerivon Cankers in inoculated shoots ( % ) 0102030405060708090100 1 year 2 year 3 year 4 year abaaaabbaa Figure 13a . Fall pruning and inoculation : Comparison within treatments of percentage of cankers from pruned shoots of 4 different ages ( 1 - to 4 - years - old ) shoots inoculated with Neofusicoccum parvum . Shoots were pruned and treated with fungicide on 29 Oct 2015 and inoculated the following day ; cankers were measured on 17 Nov 2016 . Figure 13b . Fall pruning and inoculation : Comparison of percentages of cankers within each of 4 different shoot - age categories ( 1 - to 4 - years - old ) inoculated with Neofusicoccum parvum for the fungicide treatment and control . Shoots were pruned and treated with fungicide on 29 Oct and inoculated the following day ; cankers were measured on 17 Nov 2016 . Fungicide trial in Butte County - calendar scheduled sprays . Two recordings were made of disease symptoms : blighted fruit and blighted ( and cankered ) current - season shoots ( Table 2 ) . They will discussed separately . Blighted walnuts were recorded on the day of harvest ( 28 September 2016 ) after shaking and before picking up . The untreated control had 38 % blighted fruit . Most fungicide treatments significantly reduced ( P = 0.05 ) the incidence of blighted fruit ( Table 2 ) . The best treatment was the experimental IL - 54111 at 15 oz with 6.3 % blighted fruit ( Trt 16 ) . Next was a mix of Ph - D ® and Tebucon ® ( Trt 14 ) with 8.5 % blighted fruit . Pristine ® ( Trt 4 ) with 9.9 % blighted fruit was next in effectiveness . All of the treatments with Merivon at various timings significantly reduced blighted fruit ( Table 3 ) . The best Merivon ® treatment was four sprays at bloom , on 5 May , 10 June , and 12 July . Keep in mind that the blighted fruit from these treatments were recorded before the postharvest sprays were applied on October 6 . Current season blighted shoots were recorded on 6 December 2016 . In this trial , about half of the fungicides significantly reduced ( P = 0.05 ) the incidence of Botryosphaeria canker and blight on current season spurs ( Table 2 ) . The untreated control had an average of 11.2 % spurs with cankers . The best treatment was Luna Experience ( Trt 8 ) with 1.7 blighted shoots . Next was Quash ® ( Trt 12 ) and IL - 54111 ( Trt 16 ) with 2.7 bighted shoots . Next in effectiveness was a mix of Fontelis + Abound ( Trt 1 ) with 3 % blighted shoots ; Regalia ® ( Trt 22 ) with 3.1 % blighted shoots ; Kenja ™ ( Trt 15 ) with 3.3 % blighted shoots . Among a number of treatments with different timings with Merivon ® , the timings predicated by the Leaf Wetness Model ( LWM ) proved most effective with 3.4 blighted shoots ( see timings in Table 3 ) . This is similar to last year’s results when the Merivon ® sprays predicated on the LWM were the most effective than Merivon ® sprayed at other timings . Most other timings that included a spray on 5 May ( Trts 2 , 3 , 4 , and 7 ) had only slightly more blighted shoots , with the exception of Trt 6 . This is probably because these treatments included an application just before a high risk infection event during 5 - 7 May ( see Fig . 14 ) . Fungicide trial in Butte County - best - timing spray . The evaluation of blighted fruit of a spray of different sets of 5 trees each time with Merivon ® suggests that the best timing spray was June 12 ( 11.2 % blight ) to the middle of the second week in July ( 14.3 % blight ) compared to 38.1 % for the unsprayed control ( Table 4 ) . The August spray had a slightly higher incidence of blighted fruit ( 15.4 % ) . Earlier ( including the bloom spray ) had higher incidences of blighted fruit . The postharvest spray was not evaluated since this spray was after the evaluation date . Evaluation of blighted shoots on 6 December showed a similar , but not identical trend ( Table 4 ) . The August 10 spray resulted in the lowest incidence of blighted shoots ( 2.4 % ) compared to 11.2 % for the unsprayed control . The July ( 3.6 % ) and June ( 3.7 % ) sprays were very similar . Earlier and later sprays had higher incidences of blighted shoots . California Walnut Board Trade name Active ingredient Class of fungicide ( FRAC number ) Abound ® Azoxystrobin ( 22.9 % ) Strobilurin ( 11 ) Badge ® X 2 Copper oxychloride ( 23.82 % ) Copper hydroxide ( 21.49 % ) Inorganic copper ( M1 ) Fontelis ™ Penthiopyrad ( 20 % ) Carboxamide ( 7 ) IL - 54111 Unknown Inspire Super ® Difenoconazole ( 8.4 % ) Cyprodinil ( 24.1 % ) DMI + Anilinopyrimidine ( 3 / 9 ) Kenja TM * Isofetamid Carboxamide ( 7 ) Luna ® Experience SC400 Fluopyram ( 17.6 % ) + Tebuconazole ( 17.6 % ) Carboxamide + DMI - Triazole ( 3 / 7 ) Luna ® Sensation 500 SC Fluopyram ( 21.4 % ) + Trifloxystrobin ( 21.4 % ) Carboxamide + Strobilurin ( 7 / 11 ) Manzate ® ProStik ™ Mancozeb ( 75 % ) Carbamate ( M1 / M3 ) Merivon ® Fluxapyroxad ( 21.6 % ) + Pyraclostrobin ( 21.6 % ) Carboxamide + Strobilurin ( 7 / 11 ) Movento ® 240 SC Spirotetramat Group 23 insecticide Ph - D ® Polyoxin D zinc salt ( 11.3 % ) Peptidyl pyrimidine nucleoside ( 19 ) Pristine ® Boscalid ( 25.2 % ) + Pyraclostrobin ( 12.8 % ) Carboxamide + Strobilurin ( 7 / 11 ) Quadris Top ™ Difenoconazole ( 11.4 % ) + Azoxystrobin ( 18.2 % ) DMI - Triazole + Strobilurin ( 3 / 11 ) Quash ® 50 WG Metaconazole ( 50 % ) DMI - Triazole ( 3 ) Regalia ® Reynoutria sachalinensis ( 5 % ) Biofungicide ( P5 ) R - 106506 SC * Pyraziflum Carboxamide ( 7 ) Serenade ® Opti Bacillus subtilis Tebucon ® 45 WP Tebuconazole ( 45 % ) DMI - Triazole ( 3 ) WFX - 16001 Octanoic ( caprylic ) acid ( 42 % ) Decanoic ( capric ) acid ( 28 % ) Fatty acids * Not registered currently for use on walnuts . Table 1 . Trade name , active ingredient , and class of fungicides used in 2016 in trials to control Botryosphaeria canker and blight of walnut . Spray dates Blighted fruit ( % ) 1 Blighted current season shoots 2 ( % ) Trt Treatment ( fungicide ( s ) ) Rate May 5 - 6 & 9 June 10 July 12 August 2 1 . Fontelis 4 + Abound 20 fl oz 12.0 fl oz Fontelis + Abound Fontelis + Abound Fontelis + Abound Fontelis + Abound 11.2 abc 3 3.0 ab 2 . Fontelis + Tebuconazole 45 DF 20 fl oz 8 oz Fontelis + Tebucon Fontelis + Tebucon Fontelis + Tebucon Fontelis + Tebucon 14.0 abc 3.9 a - e 3 . Fontelis + Tebuconazole Quadris Top 20 fl oz 8 oz 14 fl oz Fontelis + Tebucon Quadris Fontelis + Tebucon Quadris Top 15.6 abc 8.6 d - h 4 . Pristine 14.5 oz Pristine Pristine Pristine Pristine 9.9 abc 3.7 a - e 5 . Merivon 6.5 fl oz Merivon Merivon Merivon Merivon 12.4 abc 5.4 a - h 6 . Quadris Top 14 fl oz Quadris Quadris Quadris Quadris 12.4 abc 3.6 a - d 7 . Inspire Super 20 fl oz Inspire Super Inspire Super Inspire Super Inspire Super 17.9 bcd 6.5 b - h 8 . Luna Experience 10 fl oz Luna Exp Luna Exp Luna Exp Luna Exp 16.3 abc 1.7 a 9 . Luna Experience Movento 240 SC 10 fl oz 9 fl oz Luna Exp Movento Luna Exp Movento Luna Exp Movento Luna Exp Movento 15.7 abc 5.2 a - h 10 . Luna Experience Serenade Opti 10 fl oz 20 oz Luna Exp Serenade Luna Exp Serenade Luna Exp Serenade Luna Exp Serenade 13.9 abc 3.6 a - e 11 . Badge + Manzate ProStick 4 lbs 2.4 lbs Badge + Manzate ProStick Badge + Manzate ProStick Badge + Manzate ProStick Badge + Manzate ProStick 11.3 abc 4.1 a - f 12 . Quash 3.5 oz Quash Quash Quash Quash 14.9 abc 2.7 ab 13 . Ph - D 6.2 oz PH - D 6.2 PH - D 6.2 PH - D 6.2 PH - D 6.2 20.3 cde 6.4 b - h 14 . Ph - D Tebucon 45 6.2 oz 4 oz PH - D 6.2 Tebucon PH - D 6.2 Tebucon PH - D 6.2 Tebucon PH - D 6.2 Tebucon 8.5 ab 4.8 a - g 15 . Kenja 17 fl oz Kenja Kenja Kenja Kenja 16.4 abc 3.3 a - c 16 . IL - 54111 15 fl oz IL - 54111 IL - 54111 IL - 54111 IL - 54111 6.3 a 2.7 ab 17 . IL - 54111 17 fl oz IL - 54111 IL - 54111 IL - 54111 IL - 54111 11.1 abc 3.6 a - d 18 . Pyraziflumid 3.38 fl oz Pyraz Pyraz Pyraz Pyraz 14.9 abc 4.0 a - e 19 . Pyraziflumid 5.08 fl oz Pyraz Pyraz Pyraz Pyraz 10.7 abc 6.2 b - h 20 . WXF - 16001 5 0.35 % WXF - 16001 WXF - 16001 WXF - 16001 WXF - 16001 15.9 abc 9.4 fgh 21 . WXF - 16001 5 0.7 % WXF - 16001 WXF - 16001 WXF - 16001 WXF - 16001 12.4 abc 6.4 b - h 22 . Regalia 4 quarts Regalia Regalia Regalia Regalia 11.4 abc 3.1 a - c 23 . Pristine + Regalia 14.5 oz 4 quarts Pristine + Regalia Pristine + Regalia Pristine + Regalia Pristine + Regalia 13.9 abc 4.8 a - g 24 . Control Untreated 38.1 f 11 . 2 h Table 2 . Efficacy of fungicides against Botryosphaeria canker and blight in a Chandler walnut orchard in Butte County in 2016 . Treatment Rate Dormant Feb . 25 Delayed dormant March 10 Blighted fruit ( % ) Blighted current season shoots 2 ( % ) 25 . Liquid Lime Sulfur 16 gal . LLS 31.2 def 6.8 b - h 26 . Liquid Lime Sulfur 16 gal . LLS 34.4 ef 10.3 g - h 27 . Liquid Lime Sulfur 8 gal . LLS LLS 37.3 f 8.8 e - h 28 . Liquid Lime Sulfur 15 gal . LLS LLS 30.8 def 7.9 c - h Bloom March 31 May 5 June 10 July 12 Post - harvest Oct . 6 Blighted fruit 1 ( % ) Blighted current season shoots 2 ( % ) 1 . Merivon 4 21.2 a 1,3 4.7 a 2,3 2 . Merivon Merivon Merivon Merivon 8.3 a 3.5 a 3 . Merivon Merivon Merivon 15.5 a 4.4 a 4 . Merivon Merivon 16.9 a 6.4 ab 5 . Merivon N.D . 4.1 a 6 . Merivon Merivon Merivon Merivon 16.7 a 6.5 ab 7 . Merivon Merivon Merivon Merivon Merivon 13.4 a 4.7 a 8 . Leaf Wetness Model - LWM Merivon on : 26 April , 23 May , and 20 June 2016 12.9 a 3.4 a 9 . Untreated control 38.1 b 11.2 b Table 2 ( cont . ) Dormant and Delayed Dormant — Liquid Lime Sulfur sprays : 1 After shaking , 100 walnut fruit for each of 5 replicated trees were recorded on 28 September 2016 . 2 100 current season shoots were recorded on 6 December 2016 . 3 Numbers followed by different letters are significantly different according to the LSD test at P = 0.05 . Statistical analysis was performed on arcsine transformed data . Values presented were back transformed from the means for the arcsine transformed data . 4 Dyne - Amic at 0.0625 % vol . / vol . was added . 5 Sprayed at 200 gallons per acre . Table 3 . Efficacy of various timings of Merivon fungicide against Botryosphaeria blight in a Chandler walnut orchard in Butte Co in 2016 . 1 After shaking , 100 walnut fruit for each of 5 replicated trees were recorded on 28 September 2016 . 2 100 current season shoots were recorded on 6 December 2016 . 3 Numbers followed by different letters are significantly different according to the LSD test at P = 0.05 . Statistical analysis was performed on arcsine transformed data . Values presented were back transformed from the means for the arcsine transformed data . 4 Merivon sprayed at 6.5 fl oz / acre with Dyne - Amic at 0.0625 % vol . / vol . Bloom April 9 May 12 June 12 July 10 August 10 Post - harvest Nov 3 Blighted fruit 1 ( % ) Blighted current season shoots 2 ( % ) 1 . Merivon 4 21.2 ab 3 4.7 a 2 . Merivon 22.2 ab 5.1 a 3 . Merivon 11.2 a 3.7 a 4 . Merivon 14.3 a 3.6 a 5 . Merivon 15.4 a 2.4 a 6 . Merivon N.D . 5 4.1 a 7 . Untreated 38.1 b 11.2 b Table 4 . Efficacy of timing of single sprays of Merivon against Botryosphaeria blight in a Chandler walnut orchard in Butte Co in 2016 . 1 After shaking , 100 walnut fruit for each of 5 replicated trees were recorded on 28 September 2016 . 2 100 current season shoots were recorded on 6 December 2016 . 3 Numbers followed by different letters are significantly different according to the LSD test at P = 0.05 . Statistical analysis was performed on arcsine transformed data . Values presented were back transformed from the means for the arcsine transformed data . 4 Merivon sprayed at 6.5 fl oz / acre with Dyne - Amic at 0.0625 % vol . / vol . 5 Not determined since this treatment was done after harvest . Fungicide trial to control anthracnose in a Serr orchard in San Benito County . The San Benito plot which had a history of anthracnose in the past was used again for a fungicide trial to manage anthracnose . Disease incidence on the leaves and fruit in the plot was recorded on 20 July 2016 and reported in Table 5 . Disease incidence was moderate on the leaves in 2016 . Marssonina juglandis , the fungus responsible for anthracnose , was consistently isolated from leaf lesions before recording . So leaf lesions were considered to be anthracnose . However , lesions on the fruit consistently yielded the bacterium Xanthomonas campestris , which causes walnut blight . So fruit lesions were considered to be walnut blight . A mixture of Ph - D + Tebucon ( Trt 15 ) was the best treatment with 0.2 % leaves with anthracnose lesions compared to 41 % for the unsprayed control . Other very effective treatments were Pyraziflumid with 0.3 % ( Trt 12 ) , Merivon with 0.4 % ( Trt 2 ) , and Luna Experience + Movento with 0.6 % ( Trt 5 ) . Because this orchard has also Botryosphaeria inoculum and blighted spurs by Botryosphaeria and / or Phomopsis which were observed in the past , in early spring 2017 , buds will be collected for the BUDMON assay . This assay will provide an evaluation of the long effect of the fungicides used in 2016 against Botryosphaeria / Phomopsis canker and blight diseases . Results from this plot for fungicide sprays applied in 2015 are shown in Figure 14 . BUDMON results are discussed below . Trt # Treatment ( fungicides ) Rate per acre Spray dates Fruit with walnut blight lesions 1 , 2 ( % ) Leaves with anthracnose lesions 1 ( % ) 1 April 5 April 28 June 1 1 . Pristine 3 14.5 oz Pristine Pristine Pristine 22.7 bc 3 2.7 ab 2 . Merivon 6.5 fl oz Merivon Merivon Merivon 12.0 ab 0.4 ab 3 . Quadris Top 14 fl oz Quadris Top Quadris Top Quadris Top 17.8 abc 5.0 b 4 . Luna Experience 10 fl oz Luna Exp Luna Exp Luna Exp 17.2 abc 1.9 ab 5 . Luna Experience Movento 240 SC 10 fl oz 9 fl oz Luna Exp Movento Luna Exp Movento Luna Exp Movento 8.9 ab 0.6 ab 6 . Luna Experience Serenade Opti 10 fl oz 20 oz Luna Exp Serenade Luna Exp Serenade Luna Exp Serenade 5.0 a 1.1 ab 7 . Badge + Manzate ProStick 4 lbs 2.4 lbs Badge + Manzate ProStick Badge + Manzate ProStick Badge + Manzate ProStick 6.8 ab 0.6 ab 8 . Quash 3.5 oz Quash Quash Quash 12.9 abc 1.1 ab 9 . Kenja 17 fl oz Kenja Kenja Kenja 7.0 ab 3.1 ab 10 . IL - 54111 15 fl oz IL - 54111 IL - 54111 IL - 54111 8.9 ab 1.6 ab 11 . IL - 54111 17 fl oz IL - 54111 IL - 54111 IL - 54111 14.8 abc 1.0 ab 12 . Pyraz 3.38 fl oz Pyraz Pyraz Pyraz 8.9 ab 0.3 a 13 . Pyraz 5.08 fl oz Pyraz Pyraz Pyraz 11.0 ab 0.4 ab 14 . Ph - D 6.2 oz PH - D 6.2 PH - D 6.2 PH - D 6.2 10.6 ab 2.6 ab 15 . Ph - D Tebucon 45 6.2 oz 4 oz PH - D 6.2 Tebucon PH - D 6.2 Tebucon PH - D 6.2 Tebucon 11.8 ab 0.2 a 16 . Control Untreate d 3 3 . 1 c 41.0 c Table 5 . Fungicides , rates per acre , dates of application , and efficacy against anthracnose of Serr walnuts ( caused by Marssonina juglandis ) and walnut blight ( caused by Xanthomonas campestris pv . juglandis ) in San Benito County – 2016 . 1 100 leaves and fruit for each of 3 replicated trees were recorded on 20 July 2016 . 2 Xanthomonas camperstris pv . juaglandis , the causal agent of walnut blight , was isolated from 80 % of the Serr walnut fruit . 3 Numbers followed by different letters are significantly different according to the LSD test at P = 0.05 . Statistical analysis was performed on arcsine transformed data . Values presented were back transformed from the means for the arcsine transformed data . 4 Dyne - Amic at 0.0625 % vol . / vol . was added . Temperature , ° C 510152025 Leaf wetness , hours 0102030405060 High RiskMedium RiskLow Risk ( 4 / 22 - 23 , 16 mm ) ( 5 / 5 - 7 , 34 mm ) ( 5 / 21 , 4 mm ) ( 617 - 18 , 11 mm ) Figure 14 . Infection events as predicted by the Leaf Wetness Model ( LWM ) in 2016 for the Chandler walnut orchard located next to the Sacramento River in Butte County . Residual activity in 2016 of fungicides sprayed in the spring of 2015 . In the San Benito county Serr walnut fungicide trial for control of anthracnose , Pristine ® and Merivon ® were the most effective at reducing the incidence of Botryosphaeria in dormant buds ( Fig . 15 ) . Only Luna ® Sensation reduced the incidence of Phomopsis in these buds ( Fig . 16 ) . BUDMON results for the Butte county Chandler walnut fungicide trial showed that a mix of Fontelis ™ + Abound ® most effectively reduced Botryosphaeria in buds ( Figure 17 ) . Next in effectiveness were Merivon ® or Pristine ® . None of the treatments significantly ( P = 0.05 ) reduced the incidence of Phomopsis in buds ( Figure 18 ) , but Luna Sensation seemed to reduce the incidence to a limited degree . Botryosphaeria was rarely recovered in the BUDMON from the Merivon ® sprays predicated by the Leaf Wetness Model ( 0.08 % - Figure18 ) compared to 30 % from the unsprayed control . This was the most effective of the various timings in the 2015 fungicide trial . Other very effective Merivon ® timings in reducing Botryosphaeria in the BUDMON assay were sprays in May , June , July ( 0.6 % ) , Bloom , May , June , July , and postharvest ( 0.6 % ) ; and May , June , July , and postharvest ( 1.1 % ) . The postharvest spray alone , or combined with a bloom spray , reduced Botryosphaeria in the buds . Only a full season spray program of Merivon ® significantly reduced Phomopsis ( 0.08 % ) in the BUDMON assay compared to 8 % for the unsprayed control ( Figure 20 ) . The BUDMON from the single sprays of Merivon ® ( best timing ) followed the results of the prior season fungicide results ( Figure 21 ) . The August 10 spray had the lowest percentage of Botryosphaeria in the BUDMON assay . The other single spray treatments , with the exception of the bloom treatment , were less effective ; but still significantly ( P = 0.05 ) reduced Botryosphaeria California Walnut Board in the BUDMON assay . None of the treatments resulted in any significant differences in controlling Phomopsis compared to the unsprayed control ( Figure 22 ) . BUDMON results from a fungicide trial in Colusa County ( Figures 23 and 24 ) , and Yuba County ( Figures 25 and 26 ) had very low levels of Botryosphaeria and Phomopsis in the unsprayed controls and none of the fungicide sprays significantly reduced levels of these two fungi in the buds . Buds with Botryosphaeria ( % ) 02468101214 Fontelis 20 ozPh - D 6.2 ozControlLuna Experience 10 ozK - Phite 3 qtsQuadris Top 14 ozBadge 4 lbs + Manzate 2.4 lbsQuash 3.5 ozLuna Sensation 7.5 ozMerivon 6.5 ozPristine 14.5 oz cabcabbcccbcbcbcbc Buds with Phomopsis ( % ) 010203040506070 Quadris Top 14 ozLuna Experience 10 ozFontelis 20 ozPh - D 6.2 ozControlK - Phite 3 qtsBadge 4 lbs + Manzate 2.4 lbsQuash 3.5 ozPristine 14.5 ozMerivon 6.5 ozLuna Sensation 7.5 oz faabcabbcdeefdefcdefbcdeabcdabcd Figure 1 5 . Effect of 2015 - applied fungicides in a Serr walnut orchard in San Benito Co . on incidence of Botryosphaeria in buds collected during the following winter ( 2016 ) . A total of 50 buds per each of 5 trees were collected on 11February 2016 and processed using the BUDMON technique . Figure 1 6 . Effect of 201 5 - applied fungicides in a Serr walnut orchard in San Benito Co . on incidence of Phomopsis in buds collected during the following winter ( 2016 ) . A total of 50 buds per each of 5 trees were collected on 11 February 2016 and processed using the BUDMON technique . Buds with Botryosphaeria ( % ) 05101520253035 ControlIndar 6 ozPh - D 6.2 ozLuna Sensation 7.5 ozPh - d 6.2 oz + Tebucon 4 ozFracture 24.4 ozQuash 3.5 ozBadge 4 lbs + Manzate 2.4 lbsQuadris Top 14 ozFontelis 20 ozFontelis 20 oz + Vintre 32 ozLuna Experience 10 ozViathon 4 pintsFontelis 20 oz + Tebuconazole 8 ozFont 20 oz + Teb 8 oz / Quadris Top 14 ozPristine 14.5 ozMerivon 6.5 ozFontelis 20 oz + Abound 12 oz gabcababfgfgdefcdecdcdcddefdefefgefgefgefg gaabcababfgb - fa - ea - dc - fc - gc - fc - gefga - da - da - eb - f Buds with Phomopsis ( % ) 05101520253035 Viathon 4 pintsLuna Experience 10 ozQuadris Top 14 ozFontelis 20 ozFontelis 20 oz + Tebuconazole 8 ozPh - D 6.2 ozFontelis 20 oz + Abound 12 ozPh - d 6.2 oz + Tebucon 4 ozFontelis 20 oz + Vintre 32 ozQuash 3.5 ozFracture 24.4 ozIndar 6 ozFont 20 oz + Teb 8 oz / Quadris Top 14 ozControlBadge 4 lbs + Manzate 2.4 lbsMerivon 6.5 ozPristine 14.5 ozLuna Sensation 7.5 oz Figure 1 7 . Effect of 201 5 - applied fungicide sprays in a Chandler walnut orchard in Butte Co . ( Chico ) on the incidence of Botryosphaeria in buds collected during the following winter ( 2016 ) . A total of 50 buds per each of 5 trees were collected on 18 Feb . 2016 and processed using the BUDMON technique . Figure 1 8 . Effect of 2015 - applied fungicide sprays in a Chandler walnut orchard in Butte Co . ( Chico ) on the incidence of Phomopsis in buds collected during the following winter ( 2016 ) . A total of 50 buds per each of 5 trees were collected on 18 Feb . 2016 and processed using the BUDMON technique . California Walnut Board Buds with Botryosphaeria ( % ) 010203040 BloomUntreated ControlBloom , PostharvestPostharvestBloom , May , June , JulyMay , June , July , PostharvestBloom , May , June , July , PostharvestMay , June , JulyLeaf Wetness Model dabababcabcd Buds with Phomopsis ( % ) 0246810 PostharvestUntreated ControlBloom , May , June , JulyMay , June , July , PostharvestLeaf Wetness Model ( 3 sprays ) BloomBloom , PostharvestMay , June , JulyBloom , May , June , July , Postharvest ababababbbbb Figure 19 . Effect of 2015 sprays of Merivon ® applied at various timings in a Chandler walnut orchard in Butte Co . ( Chico ) on the incidence of Botryosphaeria in buds collected during the following winter ( 2016 ) . A total of 50 buds per each of 5 trees were collected on 18 Feb . 2016 and processed using the BUDMON technique . Figure 20 . Effect of 2015 sprays of Merivon ® applied at various timings in a Chandler walnut orchard in Butte Co . ( Chico ) on the incidence of Phomopsis in buds collected during the following winter ( 2016 ) . A total of 50 buds per each of 5 trees were collected on 18 Feb . 2016 and processed using the BUDMON technique . April 9May 12June 12July 10August 10Nov 3Unsprayed Buds with Botryosphaeria ( % ) 010203040 cababbcab April 9May 12June 12July 10August 10Nov 3Unsprayed Buds with Phomopsis ( % ) 0246810 aaaaaaa Figure 21 . Effect of 2015 single sprays of Merivon ® in a Chandler walnut orchard in Butte Co . ( Chico ) on the incidence of Botryosphaeria in buds collected during the following winter ( 2016 ) . A total of 50 buds per each of 5 trees were collected on 18 Feb . 2016 and processed using the BUDMON technique . Figure 22 . Effect of 2015 single sprays of Merivon ® in a Chandler walnut orchard in Butte Co . ( Chico ) on the incidence of Phomopsis in buds collected during the following winter ( 2016 ) . A total of 50 buds per each of 5 trees were collected on 18 Feb . 2016 and processed using the BUDMON technique . California Walnut Board Buds with Botryosphaeria ( % ) 0.00.20.40.60.81.0 ControlMerivonFontelis + TebuconLuna ExperienceLuna Experience + PostharvestQuadris Top + PostharvestQuadris TopMerivon + PostharvestFontelis + Tebucon + Postharvest aaaaaaaaa Buds with Phomopsis ( % ) 0.00.20.40.60.81.0 MerivonLuna ExperienceQuadris Top + PostharvestFontelis + TebuconControlQuadris TopMerivon + PostharvestLuna Experience + PostharvestFontelis + Tebucon + Postharvest aaaaaaaaa Figure 2 3 . Effect of 2015 - applied fungicide sprays in a Chandler walnut orchard in Colusa Co . on the incidence of Botryosphaeria in buds collected during the following winter ( 2016 ) . A total of 50 buds per replicate tree row were collected on 25 Feb . 2016 and processed using the BUDMON technique . Figure 2 4 . Effect of 2015 - applied fungicide sprays in a Chandler walnut orchard in Colusa Co . on the incidence of Phomopsis in buds collected during the following winter ( 2016 ) . A total of 50 buds per replicate tree row were collected on 25 Feb . 2016 and processed using the BUDMON technique . Buds with Botryosphaeria ( % ) 0.00.20.40.60.81.0 April / May / JuneUntreated ControlMay / June / JulyApril / May / June / July ( 0 ) aaaa Buds with Phomopsis ( % ) 0.00.20.40.60.81.0 Untreated ControlApril / May / JuneMay / June / JulyApril / May / June / July aaaaa ( 0 ) a Figure 25 . Effect of various timings of Merivon ® in 2015 in a walnut orchard in Yuba Co . on the incidence of Botryosphaeria in buds collected during the following winter ( 2016 ) . A total of 50 buds per replicate tree row were collected on 25 Feb . 2016 and processed using the BUDMON technique . Figure 26 . Effect of various timings of Merivon ® in 2015 in a walnut orchard in Yuba Co . on the incidence of Phomopsis in buds collected during the following winter ( 2016 ) . A total of 50 buds per replicate tree row were collected on 25 Feb . 2016 and processed using the BUDMON technique . LITERATURE CITED Chen , S . - F . , Hasey , J . K . , Anderson , K . , and Michailides , T . J . 2014 . Phylogeny , morphology , distribution , and pathogenicity of Botryosphaeriaceae and Diaporthaceae from English walnut in California . Plant Disease 98 : 636 - 652 . Luo , Y . , Gu , S . , Felts , D . , Puckett , R . D . , Morgan , D . P . and Michailides , T . J . 2017 . Development of qPCR systems to quantify shoot infections by canker - causing pathogens in stone fruits and nut crops . Journal of Applied Microbiology ( in press ) . Morgan , D . P . Driever , G.P . F . , Felts , D . , Krueger , W . H . , and T . J . Michailides . 2010 . Evaluation of two disease systems for Botryosphaeria panicle and shoot blight of California pistachio and efficient control based on early - season sprays . Plant Disease 93 : 1175 - 1181 . California Walnut Board California Walnut Board