Effects of Temperature on Development of Canker-Causing Pathogens in Almond and Prune.

Between 2000 and 2020, canker diseases of nut and stone fruit trees have become very widespread and severe in California. This study determined the effects of temperature on the development of canker-causing pathogens of almond and prune. Five pathogen taxa, Botryosphaeria dothidea, Cytospora leucostoma, Diaporthe (Phomopsis) neotheicola, Lasiodiplodia citricola, and Neofusicoccum mediterraneum, were used. Colony growth on medium and canker lesion development on detached shoots were measured at 10, 15, 20, 25, 30, and 35°C. The effects of temperature on colony growth differed among different pathogen taxa, although 25°C was the optimal temperature for most of the pathogens tested. The patterns of lesion growth as response to temperature were different among the different pathogens and tree crops. On almond, the highest growth rates appeared at 30°C for B. dothidea and L. citricola, but at 20°C for N. mediterraneum. The growth rates for C. leucostoma were lower than those of the other three pathogen taxa, with the highest rates recorded at 25°C. However, on prune, C. leucostoma showed greater lesion growth rates at different temperatures than the other pathogen taxa and maximum growth at 30 to 35°C. Similar trends were observed for L. citricola. The growth rates of B. dothidea and N. mediterraneum were comparatively lower than those of C. leucostoma and L. citricola.

Inoculum quantification of canker-causing pathogens in prune and walnut orchards using real-time PCR.

AIMS: A real-time quantitative PCR (qPCR) assay was established to quantify the inoculum densities in the air and rainwater for six canker-causing pathogen groups in prune and walnut orchards in California. METHODS AND RESULTS: The previously published DNA primers to target six pathogen groups including Botryosphaeria dothidea, Cytospora spp., Diplodia spp., Lasiodiplodia spp., Neofusicoccum spp. and Phomopsis spp. were used in a qPCR assay. Air samples from Burkard spore traps and rain samples from special rain collector devices were collected periodically from various prune and walnut orchards. Using the qPCR approach, we were able to quantify the concentrations of these pathogen groups in rainwater and air samples and study the dynamics of pathogen inoculum in orchards showing severe canker potential. Phomopsis spp. and Diplodia spp. were not found in all rain samples in prune orchards, although they were detected in the 2016 in the walnut orchard. The other four pathogen groups were quantified at varying concentrations in the prune and walnut orchards. Cytospora spp. in some cases showed higher concentrations in the rainwater in prune orchards. CONCLUSIONS: The rainy season during winter and early spring is a highly risky period of time for infection by the pathogens when the inoculum of these pathogens can easily spread by air and rain water, thus serving as an important inoculum source for disease initiation. The different studied pathogen groups showed different concentrations during the growing season, indicating the complexity of the components of canker-causing species in various tree crops. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed the applicability of the qPCR assay in the quantification of inoculum in tree orchards to help reveal the mechanisms of canker disease epidemics and to help design disease management strategies.

Using Allele-specific PCR for detecting multiple amino acid substitutions associated with SDHI resistance in Alternaria alternata causing Alternaria late blight in pistachio.

Alternaria late blight caused by Alternaria alternata is a major disease affecting pistachios grown in California and to some degree those grown in Arizona. Alternaria alternata is prone to quick fungicide resistance selection when single-mode of action fungicides are used. For the specific detection of five possible amino acid alterations associated with Alternaria alternata resistance to succinate dehydrogenase inhibitors used in California and Arizona pistachio orchards, we have designed five primer sets to be used as an allele-specific PCR assay (AS-PCR). Within a couple of hours from DNA extraction, the new specific-primers amplify the different PCR product sizes, according to the chosen target, allowing their differentiation on gel agarose. In our study, the AS-PCR assay consistently detected the mutations H277L and H277R at the AaSdhB gene, the mutations H134R and S135R at the AaSdhC gene, and the mutation D123E at the AaSdhD gene from A. alternata isolates collected from pistachio orchards in California and Arizona. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a rapid and cost-effective way to identify five different genotypes associated with Alternaria alternata resistance to succinate dehydrogenase inhibitors fungicides, used to control Alternaria late blight in pistachio. With the allele-specific method developed here, users will be able to identify genotypes with nucleotide substitutions which lead to a reduced sensitivity or resistance selection using a one-step PCR assay, without the use of restriction enzymes which elevates the reaction costs and the chance for errors. In addition, this study formally includes the identification and sequence accession for SdhB-H277L and SdhC-S135R amino acid substitution in A. alternata sampled from California and Arizona pistachio orchards.

Development of qPCR systems to quantify shoot infections by canker-causing pathogens in stone fruits and nut crops.

AIMS: To develop real-time PCR assays for quantification of shoot infection levels of canker disease of stone fruits and nut crops caused by six fungal pathogen groups. METHODS AND RESULTS: This study focused on six major canker-causing fungal pathogen groups: Phomopsis sp., Botryosphaeria dothidea, Lasiodiplodia sp., Cytospora sp., Neofusicoccum sp. and Diplodia sp., occurring in stone fruits and nut crops in California. DNA primers were designed to specifically target each of the six pathogen groups after the specificity tests using canker-causing and non-canker-causing pathogens and by using DNA sequences of other species from GenBank using blast. The quantitative real-time PCR (qPCR) systems were developed and used to quantify the infection levels of inoculated dried plum shoots. CONCLUSIONS: For Neofusicoccum sp. and Phomopsis sp., which were used in inoculation of walnut shoots, the values of the molecular severity ranged from 5·60 to 6·94 during the 16 days of latent infection period. The qPCR assays were more efficient, accurate and precise to quantify latent infections caused by canker-causing pathogens as compared to the traditional plating methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the potential of using the developed qPCR systems for epidemiological studies on canker diseases of woody plants.

Effects of Wound Size, Amount of Sap, and Number of Blighted Nuts on Infection of Pistachio Organs by Neofusicoccum mediterraneum.

Laboratory and field studies were conducted to determine the effects of wounding of nut exocarp, susceptibility period after wounding, and sap nut on infection of pistachio nut by Neofusicoccum mediterraneum, the main causal agent of panicle and shoot blight of pistachio. Under controlled conditions and in the field, detached nuts were inoculated with a conidial suspension 30 min before or after wounding. In addition, a 30-µl drop of pistachio sap was placed on the surface of noninjured nuts 30 min before or after they were wounded and then inoculated. Wounding increased the disease severity under both controlled and field conditions. The addition of sap increased the susceptibility of nuts under controlled conditions but not in the field, possibly due to dried sap blocking the pathogen infection. When nuts of Kerman, Kalehghouchi, and Golden Hills pistachio were wounded and inoculated at different time periods after wounding; the nuts of the three cultivars were highly susceptible to pathogen infection during at least the first 24 h after wounding. Under field conditions, there was not a clear effect of increasing the number of inoculated nuts per panicle or the inoculation position (basal or apical) in killing (blight) of the panicle. Conversely, inoculations conducted with mycelial plugs resulted in higher disease, increased the proportion of dead panicles, and resulted in faster symptom expression than inoculations conducted with a conidial suspension. To determine the temporal infection pattern, leaves and panicles were regularly collected from different orchards from 2004 to 2007 and the pathogen was isolated on medium. Important differences in latent infection were detected between years and orchards, with nut and rachis being, in general, the tissues most susceptible to infection. Results of this study help in better understanding the dynamic of infection and colonization of pistachio by N. mediterraneum.

Botrytis californica, a new cryptic species in the B. cinerea species complex causing gray mold in blueberries and table grapes.

The Botrytis cinerea species complex comprises two cryptic species, originally referred to Group I and Group II based on Bc-hch gene RFLP haplotyping. Group I was described as a new cryptic species B. pseudocinerea During a survey of Botrytis spp. causing gray mold in blueberries and table grapes in the Central Valley of California, six isolates, three from blueberries and three from table grapes, were placed in Group I but had a distinct morphological character with conidiophores significantly longer than those of B. cinerea and B. pseudocinerea We compared these with B. cinerea and B. pseudocinerea by examining morphological and physiological characters, sensitivity to fenhexamid and phylogenetic analysis inferred from sequences of three nuclear genes. Phylogenetic analysis with the three partial gene sequences encoding glyceraldehyde-3-phosate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60) and DNA-dependent RNA polymerase subunit II (RPB2) supported the proposal of a new Botrytis species, B. californica, which is closely related genetically to B. cinerea, B. pseudocinerea and B. sinoviticola, all known as causal agents of gray mold of grapes. Botrytis californica caused decay on blueberry and table grape fruit inoculated with the fungus. This study suggests that B. californica is a cryptic species sympatric with B. cinerea on blueberries and table grapes in California.

Mucor Rot-An Emerging Postharvest Disease of Mandarin Fruit Caused by Mucor piriformis and other Mucor spp. in California.

In recent years, an emerging, undescribed postharvest disease was observed on mandarin fruit after extended storage in California. We collected decayed mandarin fruit from three citrus packinghouses in the Central Valley of California in 2015 and identified this disease as Mucor rot caused by Mucor spp. Mucor rot occurred in 11 of the 15 grower lots sampled, and the percentage of Mucor rot in the total decayed fruit varied among affected grower lots, ranging from 3.3 to 93.1% with an average of 49.2%. In total, 197 isolates of Mucor spp. were obtained from decayed mandarin fruit and identified based on internal transcribed spacer sequence and morphological characteristics. Of the 197 isolates, 182 (92.4%) were identified as Mucor piriformis, 7 (3.6%) were M. circinelloides (6 M. circinelloides f. lusitanicus and 1 M. circinelloides f. circinelloides), 4 (2%) were M. racemosus f. racemosus, 3 (1.5%) were M. hiemalis, and 1 (0.5%) was M. mucedo. All species grew at 0 and 5°C, except M. circinelloides, which did not grow at 0°C. Mycelial growth was arrested at 27°C for M. piriformis; 35°C for M. racemosus f. racemosus, M. circinelloides f. lusitanicus, M. hiemalis and M. mucedo; and 37°C for M. circinelloides f. circinelloides. Optimal mycelial growth occurred at 20°C for M. piriformis and M. mucedo, 25°C for M. racemosus f. racemosus and M. hiemalis, 27°C for M. circinelloides f. lusitanicus, and 30°C for M. circinelloides f. circinelloides. M. piriformis grew significantly faster than the other four species at 5 and 20°C, and M. mucedo was the slowest in growth among the five species. Sporangiospores of M. piriformis, M. racemosus f. racemosus, and M. hiemalis germinated at both 5 and 20°C. M. circinelloides germinated at 20°C but did not germinate at 5°C after incubation for 48 h. All five Mucor spp. caused decay on mandarin fruit inoculated with the fungi, and the lesion size caused by M. piriformis was significantly larger than that caused by other species at both 5 and 20°C. Our results indicated that Mucor rot in mandarin fruit in California is caused by Mucor spp. consisting of M. piriformis, M. circinelloides, M. racemosus f. racemosus, M. hiemalis, and M. mucedo, with M. piriformis being the dominant and most virulent species. Previously, M. racemosus was reported on citrus. This is the first report of Mucor rot in citrus caused by M. piriformis, M. circinelloides, M. hiemalis, and M. mucedo.

Fungicide Resistance Profiling in Botrytis cinerea Populations from Blueberry in California and Washington and Their Impact on Control of Gray Mold.

Gray mold caused by Botrytis cinerea is a major postharvest disease of blueberry grown in the Central Valley of California and western Washington State. Sensitivities to boscalid, cyprodinil, fenhexamid, fludioxonil, and pyraclostrobin, representing five different fungicide classes, were examined for 249 (California) and 106 (Washington) B. cinerea isolates recovered from decayed blueberry fruit or flowers. In California and Washington, 7 and 17 fungicide-resistant phenotypes, respectively, were detected: 66 and 49% of the isolates were resistant to boscalid, 20 and 29% were moderately resistant to cyprodinil, 29 and 29% were resistant to fenhexamid, and 66 and 55% were resistant to pyraclostrobin. All isolates from California were sensitive to fludioxonil, whereas 70% of the isolates from Washington showed reduced sensitivity to fludioxonil. In California, 26 and 30% of the isolates were resistant to two and three classes of fungicides, respectively. In Washington, 31, 14, 16, and 9% of the isolates were resistant to two, three, four, and five classes of fungicides, respectively. Inherent risk of the development of resistance to quinone outside inhibitor (QoI) fungicides was assessed by detecting the presence of the Bcbi-143/144 intron in gene cytb. The intron was detected in 11.8 and 40% of the isolates in California and Washington, respectively, suggesting that the risk of QoI resistance is higher in California than in Washington. On detached blueberry fruit inoculated with 11 isolates exhibiting different fungicide-resistant phenotypes, most fungicides failed to control gray mold on fruit inoculated with the respective resistant phenotypes but the mixture of cyprodinil and fludioxonil was effective against all fungicide-resistant phenotypes tested. Our findings would be useful in designing and implementing fungicide resistance management spray programs for control of gray mold in blueberry.

Intraspecific Diversity of Monilinia fructicola and M. laxa Populations from Blossoms and Fruit of Different Hosts in Greece.

The genetic variation among 145 isolates of Monilinia fructicola and 156 isolates of M. laxa collected from two distinct regions of Greece (Imathia and Larissa) was analyzed using intersimple-sequence repeat markers. The Monilinia spp. isolates had been collected from infected fruit or blossoms of peach, apricot, sweet cherry, and plum. Calculation of Nei's gene diversity and Shannon's diversity indices showed that M. fructicola populations had higher genetic diversity compared with M. laxa populations in both regions sampled. The levels of genetic diversity were similar between populations obtained from diseased blossoms and fruit for each species and the main variances were all from within rather than between populations for the respective regions, hosts, and organ of origin. Genetic distance (Nei's analysis) was lower between peach and apricot populations than between cherry and plum populations of M. fructicola. M. fructicola isolates from peach and apricot and from sweet cherry and plum were clustered together, while M. laxa isolates clustering based on the host of origin was not possible. The analysis of index of association showed the absence of sexual recombination for both species. The derived data support the hypothesis of a long presence of M. fructicola in Greece, and provide evidence of specialization of M. fructicola populations based on their host of origin.

First Report of Botrytis pseudocinerea Causing Gray Mold on Blueberry in North America.

Botrytis cinerea has previously been shown to consist of two sibling species, referred to as Group I and Group II, that can be differentiated by PCR-RFLP analysis of the Bc-hch gene, a vegetative incompatibility locus (1). Group I has recently been described as a new cryptic species, B. pseudocinerea (4). Gray mold caused by B. cinerea is a major postharvest disease of blueberries in the Central Valley of California. In 2012 and 2013, blueberry fruit were sampled at harvest from various locations in the region and stored at 0 to 1°C for 5 weeks, and fungi were isolated from decayed fruit. In total, 526 isolates of Botrytis spp. were obtained. Genomic DNA was extracted and PCR-RFLP of a fragment of the Bc-hch gene was performed. Four isolates showed the distinctive restriction band pattern associated with Group I (1). The identity of these four isolates was further investigated by sequencing portions of four genes: internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPBII), using the primers described previously (3,4). Sequences were deposited in GenBank (Accession Nos. KJ796643 to 58). BLAST analysis showed that sequences of all four genes for the four isolates were 99.8 to 100% similar to those of B. pseudocinerea. Morphological characteristics of the four blueberry isolates were examined as described previously (4). On potato dextrose agar, colonies were gray; the mycelial growth rate was 26 mm/day at 19°C in the dark. Conidiophores were simple and erect, and conidia were borne in grapelike clusters, one celled, hyaline, elliptical to ovoid, 6.5 to 15.7 × 5.6 to 9.8 µm (average of 7.4 × 10.1 µm). As reported previously, none of the morphological characters was able to differentiate between B. cinerea and B. pseudocinerea (4). To test pathogenicity, freshly harvested organic blueberry fruits were treated with 0.5% sodium hypochlorite for 2 min, rinsed with sterile water, wounded using a sterile needle, and inoculated by placing 1 µl of a conidial suspension (1.0 × 105 spores/ml) from each isolate into the wound with a pipette. Inoculated fruit (10 for each isolate) were incubated at 20°C for 5 days in the dark. Experiments were performed twice. All inoculated fruit developed rot, and no decay was observed on the noninoculated controls. All four isolates of B. pseudocinerea were pathogenic, and the fungus was re-isolated from decayed fruit. B. pseudocinerea isolates are known to be naturally insensitive to fenhexamid (1,4). Sensitivity of the four isolates to fenhexamid was examined in vitro as previously described (4). The EC50 values for fenhexamid for the four isolates ranged from 7.7 to 9.9 µg/ml and isolates were considered resistant to fenhexamid (1,4). Based on the morphological, physiological, and genetic characteristics, the four blueberry isolates were identified as B. pseudocinerea. It appeared that this species was present at very low frequency (0.76%) in blueberry fields in California. Previously, B. pseudocinerea has been reported from French, German, and New Zealand vineyards (1,2,4). To our knowledge, this is the first report of B. pseudocinerea causing gray mold in blueberry in California and in North America. References: (1) E. Fournier et al. Mycologia 95:251, 2003. (2) P. R. Johnston et al. Plant Pathol. 63:888, 2014. (3) M. Staats et al. Mol. Biol. Evol. 22: 333, 2005. (4) A.-S. Walker et al. Phytopathology 101:1433, 2011.

Sensitivities of Baseline Isolates and Boscalid-Resistant Mutants of Alternaria alternata from Pistachio to Fluopyram, Penthiopyrad, and Fluxapyroxad.

Resistance of Alternaria alternata to boscalid, the first succinate dehydrogenase inhibitor (SDHI) fungicide labeled on pistachio, has become a common occurrence in California pistachio orchards and affects the performance of this fungicide. In this study, we established the baseline sensitivities of A. alternata to the new SDHIs fluopyram, fluxapyroxad, and penthiopyrad and assessed their cross resistance patterns with boscalid. Examination of the effective fungicide concentration that inhibits mycelial growth to 50% relative to the control (EC50) for 50 baseline isolates revealed that the majority were sensitive to boscalid, penthiopyrad, fluopyram, and fluxapyroxad. Analysis of EC50 values for boscalid for 117 A. alternata isolates originating from boscalid-exposed orchards showed that 44, 3, 1, and 69 isolates had sensitive, reduced sensitivity, moderately resistant, and highly resistant boscalid phenotypes, respectively. Molecular investigation of the occurrence of known SDH mutations showed that, among the 69 isolates highly resistant to boscalid, 44, 2, 14, and 1 isolates possessed the mutations leading to the H277Y, H277R, H134R, and H133R amino acid substitutions in AaSDHB, AaSDHB, AaSDHC, and AaSDHD subunits, respectively. Some SDHB or SDHC mutants displayed highly sensitive, sensitive, or reduced sensitivity phenotypes toward penthiopyrad or fluxapyroxad, whereas other had low, moderate, or high levels of resistance to these fungicides. In contrast, all the SDHB mutants were sensitive to fluopyram, while 10, 5, and 1 SDHC mutants had sensitive, reduced sensitivity, and moderately resistant fluopyram phenotypes, respectively. The SDHD mutant had reduced sensitivity to fluopyram and penthiopyrad but was highly resistant to fluxapyroxad. The discrepancies of cross-resistance patterns between SDHIs suggest that their binding sites in complex II may differ slightly and that additional mechanisms of resistance to these compounds are likely involved. Ultimately, the findings of this study should lead to the rational and sustained deployment of new SDHIs in Alternaria late blight spray programs.

First Report of Lasiodiplodia theobromae Associated with Stem Canker of Almond in California.

California is a major almond (Prunus dulcis) producer in the world. In September 2012, 2-year-old almond trees from an orchard in Fresno Co. with stem cankers were submitted for disease diagnosis. In a survey of the orchard, 12 ha (1,500 Nonpareil and 1,800 Monterey almond trees) of 48 ha trees had been killed apparently due to a stem canker. The cankers developed above the graft union, were covered with amber sap, and often girdled the trunk. Isolations made from tissues at the canker margins onto acidified potato dextrose agar (PDA) yielded two fungi, Macrophomina phaseolina (Tassi) Goid and Lasiodiplodia theobromae (Pat.) Griffon & Maubl (1). M. phaseolina and L. theobromae were isolated from eight and two of 10 cankered trees, respectively. No mixed infections were found. M. phaseolina isolates were characterized by gray hyphae that turned black with developing microsclerotia. L. theobromae isolates were characterized by white, aerial mycelium that turned mouse gray after 5 days. Young conidia were ellipsoidal, thick walled, initially hyaline, granular, and nonseptate; aged conidia were brown, 1-septate with longitudinal striations in the wall. Identity was confirmed by analyses of the internal transcribed spacer (ITS), ß-tubulin 2 (BT2), and the translation elongation factor 1-alpha (TEF-1α) gene regions. BLAST searches at GenBank showed a high identity with reference sequences of type specimens both for M. phaseolina (isolates 7E64 to 7E69: ITS, 100%; BT2, 99%; TEF-1α, 99%) and L. theobromae (isolates 7E86 to 7E88: ITS, 99%; BT2, 99%; TEF-1α, 100%). Sequences of three gene regions were deposited as GenBank accessions KC357271 to KC357279 (ITS); KC357280 to KC357288 (BT2); and KC357289 to KC357297 (TEF-1α). The pathogenicity of M. phaseolina and L. theobromae to P. dulcis cultivars Butte, Carmel, Nonpareil, and Padre was investigated in an orchard at KARE using four isolates of M. phaseolina (7E64, 7E65, 7E66, and 7E69) and two isolates of L. theobromae (7E86 and 7E88). Ten 2-year-old branches per isolate from 7-year-old trees were inoculated with each isolate in late September 2012, after removing the bark with a 7-mm cork borer and placing a 7-day-old 7-mm-diameter agar plug bearing mycelium of each isolate directly into the fresh wound, mycelium side down. Ten additional branches of each of the four cultivars were inoculated with sterile PDA plugs and served as negative controls. Three weeks after inoculation, the average lesion produced by M. phaseolina on Butte, Carmel, Nonpareil, and Padre was 53, 52, 41, and 37 mm in length, respectively. Lesions produced by L. theobromae were 191, 206, 194, and 103 mm in length on the four cultivars, respectively. No disease lesion, only wounds, were produced on negative controls. Lesions produced by both pathogens were longer (P < 0.05) than wounds on the controls (average length 10 mm on all cultivars). Both L. theobromae isolates killed branches of cultivars Butte, Carmel, and Nonpareil in 2 weeks. M. phaseolina and L. theobromae were reisolated from the inoculated branches, and no fungus was reisolated from controls. Based on pathogenicity results, L. theobromae is more virulent to almond branches than M. phaseolina. To our knowledge, this is the second report of M. phaseolina (2) and the first report of L. theobromae as pathogens of P. dulcis trees in California. References: (1) A. Alves et al. Fungal Diversity 28:1, 2008. (2) P. Inderbitzin et al. Mycologia 102:1350, 2010.

First Report of Lasiodiplodia citricola and Neoscytalidium dimidiatum Causing Death of Graft Union of English Walnut in California.

California produces 99% of the English walnuts (Juglans regia) in the USA. In August 2012 in Tulare County, about 5,000 out of 90,000 trees were killed in a walnut nursery by a distinct black canker that developed around the graft union. The cankers appeared to be initiated at the heading cut on the rootstock, and expanded down to the rootstock and through the budded union up to the scion, resulting in mortality of scion. The walnut nursery was located adjacent to a mature walnut orchard. The fungi isolated from the cankers were identified as Lasiodiplodia citricola and Neoscytalidium dimidiatum based on morphological characteristics and DNA sequence comparisons. L. citricola was isolated from one of the 10 graft unions, while N. dimidiatum from the other nine. L. citricola isolates were characterized by white, aerial mycelium on potato dextrose agar that turned gray after 4 days and produced ellipsoidal to ovoid hyaline one-celled conidia that became 2-celled and brown with thick walls and longitudinal striations in the wall (1). N. dimidiatum isolates were characterized by ellipsoid to ovoid, hyaline conidia with a truncate base and an acutely rounded apex, initially 1-celled, and some becoming brown and 2-celled at maturity; no muriform conidia were produced (3). These identifications were confirmed by analyses of the ITS, BT2, and TEF-1α gene regions. The three gene regions were amplified by using the primers and methods described in (4). For L. citricola (isolates 7E78 to 7E80), a DNA sequence BLASTn at GenBank showed 100% identity with accessions GU945354 (ITS) and GU945340 (TEF-1α) of the ex-type specimen (CBS124707, BT2 sequencing data was not available) (3). For N. dimidiatum (isolates 7E61 to 7E63), a BLASTn search showed a high identify (ITS, 100%; BT2, 99%; TEF-1α, 99%) with reference sequence of N. dimidiatum (ITS, GQ330903; BT2, GU251768; TEF-1α, GU251240). Sequences of the studied DNA regions were deposited to GenBank as KC357298 to KC357303 (ITS); KC357304 to KC357309 (BT2); and, KC357310 to KC357315 (TEF-1α). The pathogenicity of L. citricola in comparison with N. dimidiatum in J. regia cvs. Chandler, Tulare, and Vina was evaluated in an orchard at KARE, by using two isolates each of L. citricola (7E78, 7E80) and N. dimidiatum (7E61, 7E63). Pathogenicity tests were performed by inoculating ten 2-year-old branches per isolate in late September 2012 by the method described in (2). After 3 weeks, the average lesion lengths caused by L. citricola on Chandler, Tulare, and Vina were 152, 156, and 188 mm, respectively, and 32, 38, and 34 mm, respectively, caused by N. dimidiatum. The lesion length averages produced on the three cultivars by the four isolates were all significantly (P < 0.05) longer than their respective controls (average length 10 mm on all cultivars). L. citricola, but not N. dimidiatum, killed branches of Chandler, Tulare, and Vina in 10 days. Both L. citricola and N. dimidiatum were reisolated from the inoculated branches and no fungus was isolated from the control, confirming Koch's postulates. These results confirmed that the walnut graft union canker was caused by either L. citricola or N. dimidiatum. To our knowledge, this is the first report of death of newly grafted walnut trees caused by L. citricola and N. dimidiatum worldwide, and also the first report of L. citricola infecting walnut worldwide. References: (1) J. Abdollahzadeh et al. Persoonia. 25:1, 2010. (2) S. F. Chen et al. Plant Dis. 97:994, 2013. (3) D. Pavlic et al. Mycologia. 106:851, 2008. (4) B. Slippers et al. Mycologia. 96:83, 2004.

First Report of Twig Canker on Willow Caused by Colletotrichum acutatum in California.

Following prolonged spring rains and cool summer weather in 2010, mature weeping willow trees (Salix babylonica L.) growing next to a manmade lake in Marin County, CA, showed symptoms of a previously undescribed disease. During summer, small branches developed dark brown to black, sunken cankers. Canker lengths ranged from 3 to 20 cm. Within the cankered areas, affected twigs, shoots, and leaves turned brown, collapsed, and died. The distal portions of infected branches also died, giving the trees a blighted appearance. Acervuli and pink sporulation were observed in the canker tissue. When placed on acidified potato dextrose agar (A-PDA), canker tissues consistently yielded one type of fungal organism. On A-PDA, isolates produced gray aerial mycelium, acervuli, and single-celled fusiform conidia. Two isolates were identified as Colletotrichum acutatum based on sequence analysis of the ITS region of the ribosomal DNA and the 1-kb intron of the glutamine synthase gene (1) and fungal morphology (2,3) (GenBank Accession Nos. JQ951597 and JQ951598). The willow isolates examined were identified as C. acutatum based on a 99% identity of the ITS sequence with accession FR716517 and a 98% identity of the 1-kb intron sequence with accession GQ387248 in GenBank. Interestingly, the isolates were confirmed to be homothallic producing perithecia from monoconidial cultures. To demonstrate Koch's postulates, inocula were prepared from 2-week-old colonies of each of four isolates grown on A-PDA. Using containerized weeping willow trees as test material, shallow slits were cut into the epidermis of small (1.5-cm diameter or less) branches; one colonized agar plug was placed within each cut area and the epidermis was resealed by wrapping the branch with Parafilm. Ten inoculations were made for each isolate and inoculated plants were maintained in a greenhouse. After 4 weeks, inoculated branches exhibited dark cankers and twig dieback. C. acutatum was reisolated from all symptomatic cankers and matched the characteristics of the original isolates. Control twigs, inoculated with sterile agar plugs, did not develop any blight symptoms. This experiment was repeated and the results were the same. To our knowledge, this is the first documentation of C. acutatum as a pathogen of weeping willow in California. The disease resulted in repeated defoliation of trees in the Santa Venetia area of Marin County. Badly infected trees declined as a result of repeated defoliation and twig loss. Discussions with parks personnel suggested that the disease may have been present at low levels in the area for some years, and that disease severity increased dramatically with weather that was atypically wet and cool (max. mean temps. 5.5°C cooler and 6 cm more total rainfall than the records of the previous two years) for the area during May and June 2010, when the disease was discovered. References: (1) J. C. Guerber et al. Mycologia 95:872, 2003. (2) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (3) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990.

Competitive Ability and Fitness of Alternaria alternata Isolates Resistant to QoI Fungicides.

Fungicides that act as quinone outside inhibitors (QoIs) constitute a fungicide group extensively used against Alternaria late blight of pistachio caused by Alternaria spp. However, developement of resistance to this fungicide class constitutes an important threat for the succesful control of the disease. This study was conducted to determine whether development of resistance to QoIs is associated with a fitness cost, by measuring several biological and epidemiological parameters and estimating the competitive ability in four QoI-resistant and four QoI-sensitive Alternaria alternata isolates. Fitness parameters measured were mycelial growth and spore production in vitro, disease latent period, aggressiveness, and spore production on detached pistachio leaves. The competitive ability of resistant isolates was assessed in coinoculation experiments with sensitive isolates on detached pistachio leaves, using a real-time polymerase chain reaction assay technique. Fitness parameters between grouped QoI-resistant and QoI-sensitive isolates were not significantly different (P = 0.13, 0.21, 0.31, and 0.27 for sporulation in vitro, mycelial growth, incubation period, and sporulation in vivo, respectively), while resistant isolates, as a group, showed a higher aggressiveness (P = 0.01) compared with the sensitive isolates. The data indicate that the resistant strains did not account for a fitness cost compared with the sensitive ones under the conditions of testing. The outcome of the competition experiments was isolate dependent. In two pairs, the resistance frequencies increased whereas, in the remaining two pairs of isolates, resistance frequency decreased, suggesting that the resistant isolates were competitive similarly to the sensitive isolates.

Quantification of conidial density of Aspergillus flavus and A. parasiticus in soil from almond orchards using real-time PCR.

AIMS: To design the Aspergillus flavus and Aspergillus parasiticus-specific primers and a real-time PCR assay for quantification of the conidial density in soil. METHODS AND RESULTS: Aspergillus flavus and A. parasiticus-specific DNA primers were designed based on internal transcribed spacer sequences to distinguish these two species and from other Aspergillus and other fungal species. A method of pathogen DNA extraction directly from soil samples was developed. Using the designed primers, a real-time PCR assay was developed to quantitatively determine the conidial density of each A. flavus and A. parasiticus in soil, after generating corresponding standard curves. Known conidial densities of each A. flavus or A. parasiticus in soil significantly correlated with those tested with the real-time PCR. CONCLUSIONS: This study demonstrated the applicability of the real-time PCR assay in studies of quantifying A. flavus and A. parasiticus in soil as inoculum sources. SIGNIFICANCE AND IMPACT OF THE STUDY: The A. flacus and A. parasitic-specific primers can be widely used in aflatoxin research. The real-time PCR assay developed in this study provides a potential approach to quantify the plant pathogen density from not only soil but also other sources in relation to aflatoxin contamination from environment, food and feed commodities.

Molecular characterization of toxigenic and atoxigenic Aspergillus flavus isolates, collected from peanut fields in China.

AIMS: The objectives of this study were to assess the genetic relationships between toxigenic and atoxigenic isolates of Aspergillus flavus collected from peanut fields in China, and to analyse deletions within the aflatoxin biosynthetic gene cluster for the atoxigenic isolates. METHODS AND RESULTS: Analysis of random-amplified polymorphic DNA and microsatellite-primed PCR data showed that the toxigenic and atoxigenic isolates of A. flavus were not clustered based on their regions and their ability of aflatoxin and sclerotial production. These results were further supported by DNA sequence of ITS, pksA and omtA genes. PCR assays showed that 24 of 35 isolates containing no detectable aflatoxins had the entire aflatoxin gene cluster. Eleven atoxigenic isolates had five different deletion patterns in the cluster. CONCLUSIONS: Toxigenic and atoxigenic isolates of A. flavus are genetically similar, but some atoxigenic isolates having deletions within the aflatoxin gene cluster can be identified readily by PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: Because the extensive deletions within the aflatoxin gene cluster are not rare in the atoxigenic isolates, analysis of deletion within the cluster would be an effective method for the rapid screening of atoxigenic isolates for developing biocontrol agents.

Characterization of mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with Boscalid resistance in Alternaria alternata from California pistachio.

Thirty-eight isolates of Alternaria alternata from pistachio orchards with a history of Pristine (pyraclostrobin + boscalid) applications and displaying high levels of resistance to boscalid fungicide (mean EC(50) values >500 microg/ml) were identified following mycelial growth tests. A cross-resistance study revealed that the same isolates were also resistant to carboxin, a known inhibitor of succinate dehydrogenase (Sdh). To determine the genetic basis of boscalid resistance in A. alternata the entire iron sulphur gene (AaSdhB) was isolated from a fungicide-sensitive isolate. The deduced amino-acid sequence showed high similarity with iron sulphur proteins (Ip) from other organisms. Comparison of AaSdhB full sequences from sensitive and resistant isolates revealed that a highly conserved histidine residue (codon CAC in sensitive isolates) was converted to either tyrosine (codon TAC, type I mutants) or arginine (codon CGC, type II mutants) at position 277. In other fungal species this residue is involved in carboxamide resistance. In this study, 10 and 5 mutants were of type I and type II respectively, while 23 other resistant isolates (type III mutants) had no mutation in the histidine codon. The point mutation detected in type I mutants was used to design a pair of allele-specific polymerase chain reaction (PCR) primers to facilitate rapid detection. A PCR-restriction fragment length polymorphism (RFLP) assay in which amplified gene fragments were digested with AciI was successfully employed for the diagnosis of type II mutants. The relevance of these modifications in A. alternata AaSdhB sequence in conferring boscalid resistance is discussed.

First Report of Stemphylium botryosum Causing Leaf Blight of Kiwi in the Province Imathia, Northern Greece.

In Greece, kiwi (Actinidia deliciosa) is mostly found in the northern part of the country where approximately 440,000 ha are grown. In the summer of 2006, a Stemphylium sp. was frequently isolated from leaves of kiwi (cv. Hayward) grown in the province of Imathia. Symptomatic leaves were covered with irregular, necrotic, brown areas. Lesions had a distinct margin that, in some cases, covered a wide part of the diseased leaves. Intense symptoms were frequently observed and associated with defoliation. This Stemphylium sp. was consistently isolated from diseased leaves onto potato dextrose agar (PDA) after surface sterilization with 0.1% chlorine solution. On the basis of morphological characteristics of mycelia, dimensions (length 20 to 29 µm and width 14 to 21 µm) and mean length/width ratio (1.42 µm) of conidia, and width and apical cell width of condiophores, the fungus was identified as Stemphylium botryosum (Wallr.) (2,3) Koch's postulates were completed in the laboratory by inoculating leaves of kiwi (cv. Hayward) with an isolate of S. botryosum originated from a symptomatic leaf of a Hayward kiwi. Twenty leaves were surface sterilized by dipping them into 0.1% chlorine solution for 2 to 3 min, washing in sterile distilled water, and allowing them to dry in a laminar flow hood. A leaf was then placed into a petri plate containing a wet, sterilized paper towel. Inoculation was made by transferring a 5-mm-diameter mycelial disc from the margins of a 7-day-old culture onto the center of each leaf surface. Petri plates were closed and incubated at 25°C with 12 h of light for 6 days. Koch's postulates were satisfied when the same S. botryosum was reisolated from 100% of inoculated leaves that developed symptoms similar to those observed in the vineyards. Leaves inoculated with a PDA plug alone (with no S. botryosum) did not develop any symptoms. Previously, Alternaria alternata was reported as the causal agent of a leaf spot pathogen of kiwi (1,4). To our knowledge, this is the first report of the occurrence of S. botryosum causing leaf blight of kiwi in Greece and worldwide. This pathogen can cause a high level of defoliation in diseased plants. References: (1) L. Corazza et al. Plant Dis. 83:487, 1999. (2) M. B. Ellis. Dematiaceous Hyphomycetes. Mycology Institute. London, England, 1971. (3) E. G. Simmons. Mycologia 61:1, 1969. (4) C. Tsahouridou and C. C. Thanassoulopoulos. Plant Dis. 84:371, 2000.

High Susceptibility of Olive Cultivar FS-17 to Alternaria alternata in Southern Spain.

Traditional olive orchards in Spain have been planted at a density of 70 to 80 trees per ha with three trunks per tree. During the last decade, the hedgerow orchard, in which planting density is approximately 2,000 trees per ha, was developed. In 2006 and 2007, we noted a severe outbreak of fruit rot in FS-17, a new cultivar from Italy, in an experimental hedgerow planting in Córdoba, southern Spain. The incidence of fruit rot in 'FS-17' was 80% in January of 2006 and 24% in January of 2007. Cvs. Arbosana, IRTA-i18 (a selected clone from 'Arbequina'), and Koroneiki had no symptoms in either year of the study. Disease incidence in 'Arbequina' was <0.1% only in 2006. Affected fruits were soft with gray-white skin and they eventually mummified. Black-green sporodochia were observed on the surface of diseased fruits. A fungus was isolated from diseased fruits on potato dextrose agar (PDA) and incubated at 22 to 26°C with a 12-h photoperiod. After 8 days of growing on PDA, fungal colonies formed conidial chains having a main axis with up to 10 conidia and secondary and tertiary short branches with two to four conidia. Conidia were obpyriform, ovoid, or ellipsoidal, without a beak or with a short beak, had up to four transverse septa, and measured 11.7 to 24.7 (mean 19.6) µm long and 7.7 to 13.0 (mean 9.6) µm wide at the broadest part of the conidium. The length of the beak of conidia was variable, ranging from 0 to 28.6 (mean 5.5) µm. The fungus was identified as Alternaria alternata (1). Pathogenicity tests were performed by spraying 40 mature fruits of 'FS-17' with a spore suspension (1 × 106 spores per ml). The same number of control fruits was treated with water. After 21 days, inoculated fruit developed symptoms that had earlier been observed in the field. A. alternata was reisolated from lesions on all infected fruits. The fungus was not isolated from any of the control fruits. The experiment was performed twice. The new growing system and the high susceptibility of some olive cultivars, such as FS-17, may result in a high incidence of disease caused by a pathogen that is generally characterized as weakly virulent. To our knowledge, this is the first report of A. alternata causing a severe outbreak of fruit rot on olive trees in the field. References: (1) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002.