Sporulation and Dispersal of the Biological Control Agent Aspergillus flavus AF36 Under Field Conditions.

Aflatoxins are carcinogens produced by the fungi Aspergillus flavus and A. parasiticus that contaminate pistachio crops. International markets reject pistachio when aflatoxins exceed permitted maximum levels. Releasing the atoxigenic strain AF36 of A. flavus is the leading aflatoxin pre-harvest control method. The product AF36 Prevail, sorghum grains coated with AF36 propagules, has been used in California since 2017. However, a high percentage of grains of the Prevail fail to sporulate in orchards. Here, the effect of soil moisture on the percentage of AF36 product grains sporulating (SG) and the quantity of spores per grain using a sporulation index (SI) was determined. Under controlled conditions, SG was higher than 85% when soil moisture was 13% or more, and SI increased with increasing soil moisture from 8.4 to 21%. The highest AF36 sporulation occurred near the micro-sprinklers when the grains were not impacted by the irrigation water drops. Arthropod predation was responsible for lost product grains, which was more pronounced in non-tilled soil than in tilled soil. Dispersal of the AF36 spores decreased markedly with the height and distance from the inoculum source, following a pattern of diffusion equations. However, AF36 spores easily reached canopies of pistachios located 10 m from the inoculum source. Our results indicate that AF36 Prevail should be applied close to the irrigation line in the moist soil area but avoiding the areas where excess irrigation causes water accumulation. The biocontrol of aflatoxins in California's pistachio production areas was optimized by improving the field realization of the biological control agent.

Reassessing the Etiology of Aspergillus Vine Canker and Summer Bunch Rot of Table Grapes in California.

Fungal taxonomy is in constant flux, and the advent of reliable DNA barcodes has enabled the enhancement of plant pathogen identification accuracy. In California, Aspergillus vine canker (AVC) and summer bunch rot (SBR) are economically important diseases that affect the wood and fruit of grapevines, respectively, and their causal agents are primarily species of black aspergilli (Aspergillus section Nigri). During the last decade, the taxonomy of this fungal group has been rearranged several times using morphological, physiological, and genetic analyses, which resulted in the incorporation of multiple cryptic species that are difficult to distinguish. Therefore, in this study, we aimed to reassess the etiology of AVC and SBR using a combination of morphological observations with phylogenetic reconstructions based on nucleotide sequences of the calmodulin (CaM) gene. Results revealed that the isolates causing AVC from recent isolations corresponded to A. tubingensis, whereas the isolates obtained from initial surveys when the disease was discovered were confirmed as A. niger and A. carbonarius. Similarly, the isolates obtained from table grapes with SBR symptoms and from spore traps placed in those vineyards were identified primarily as A. tubingensis, followed by A. niger and A. carbonarius. Notably, the A. niger isolates formed a subclade with strains previously known as A. welwitschiae, which is a species that was recently synonymized with A. niger. Overall, the most prevalent species was A. tubingensis, which was associated with both AVC and SBR, and representative isolates recovered from AVC-symptomatic wood, berries SBR symptoms, and spore traps were equally pathogenic in healthy wood and berries of 'Red Globe' grapevines. This study also constitutes the first report of A. tubingensis causing AVC and SBR of grapes in California and in the United States.

Survey on Latent Infection of Canker-Causing Pathogens in Budwood and Young Trees from Almond and Prune Nurseries in California.

Almond band canker and prune Cytospora canker have become more severe in the last decade, especially in young orchards, in California. To test our hypothesis that young trees from nurseries could carry the canker-causing pathogens at latency phase to new orchards through transplanting, a multiyear survey on latent infection of canker-causing pathogens of budwood and young trees of almond and prune nurseries in California was conducted. A total of more than 1,730 samples including shoots of rootstocks and scions and grafting union cuttings were collected from 11 nurseries. A real-time quantitative PCR assay was applied to quantify the latent infection levels by six canker-causing pathogen taxa: Botryosphaeria dothidea and species of Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum, and Phomopsis. For almond, the average incidences of latent infection caused by Lasiodiplodia spp. (43.6%) and Neofusicoccum spp. (24.2%) were significantly greater than those by the other four pathogen taxa. The molecular severity (MS) of latent infection caused by Neofusicoccum spp. (3.6) was significantly greater than those caused by other pathogen taxa, except for Lasiodiplodia spp. (2.6). For prune, the average incidence of latent infection caused by Cytospora spp. (13.5%) was significantly higher than those caused by B. dothidea (1.5%) and Diplodia spp. (1.3%) but not significantly higher than those caused by Lasiodiplodia spp. (6.9%), Neofusicoccum spp. (6.3%), and Phomopsis spp. (7.7%), respectively. Moreover, the average MS values caused by Cytospora spp. (3.8) and Neofusicoccum spp. (3.2) were the highest followed by those caused by B. dothidea (1.4), Lasiodiplodia spp. (2.2), and Phomopsis spp. (2.3). Different almond varieties showed various levels of susceptibilities to different canker-causing pathogens. This study concluded that Lasiodiplodia and Neofusicoccum are the predominant pathogen species in almond, and Cytospora is the most important canker-causing pathogen species in prune in nurseries. These findings confirmed the observations of predominancy of canker-causing pathogens in almond and prune orchards in California.

Ochratoxin A Contamination of California Pistachios and Identification of Causal Agents.

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium spp., which contaminates many crops, including pistachios. Pistachios contaminated with OTA may be subjected to border rejections resulting in significant economic losses to the United States agricultural revenues. The current study examined prevalence of OTA in California-grown pistachios and identified its causal agents. OTA was detected in 20% of samples from 2018 to 2021 (n = 809), with 18% of samples exceeding the European Union regulatory limit of 5 µg/kg. Fungi potentially responsible for OTA contamination were isolated from leaves, nuts, and soil collected from 14 pistachio orchards across California. A total of 1,882 isolates of Aspergillus section Nigri and 85 isolates of section Circumdati were recovered. Within section Nigri, 216 (11.5%) isolates were identified as potential OTA producers using a boscalid-resistance assay. Phylogenetic analyses of partial gene sequences for ß-tubulin and calmodulin genes resolved section Circumdati into four species: A. ochraceus (33%), A. melleus (28%), A. bridgeri (21%), and A. westerdijkiae (19%). A. westerdijkiae produced the highest levels of OTA in inoculated pistachios (47 µg/g), followed by A. ochraceus (9.6 µg/g) and A. melleus (3.3 µg/g). A. bridgeri did not produce OTA. OTA production by section Circumdati was optimal from 20 to 30°C. All 216 boscalid-resistant isolates from section Nigri were identified as A. tubingensis, and representative isolates (n = 130) produced 3.8 µg/kg OTA in inoculated pistachios. This is the first detailed report on OTA contamination and causal fungi in California pistachios and will be helpful in devising effective management strategies.

Prevalence, Identity, Pathogenicity, and Infection Dynamics of Botryosphaeriaceae Causing Avocado Branch Canker in California.

Botryosphaeria branch canker and dieback of avocado (Persea americana Mill.) has expanded in avocado-growing areas in recent years. Twenty-one avocado groves in the major producing regions of California were surveyed in 2018 and 2019. Monthly inoculations of wounded, green, and lignified branches of 'Hass' and 'Lamb Hass' were conducted. Botryosphaeriaceae were the predominant fungi recovered from cankered tissues collected across the surveyed traditional and high-density orchards and caused symptoms on all six sampled cultivars. These fungi were also recovered in asymptomatic twigs and other organs and thus exist as a potential reservoir for future infections. Molecular analyses of 173 isolates showed that Neofusicoccum luteum had the greatest incidences across sites and cultivars, with 83 and 29% recovered from Hass and Lamb Hass, respectively. Pathogenicity tests on excised (Hass, GEM, and Hass mutants) and attached shoots from potted (Hass) and mature avocado trees (Hass and Lamb Hass) showed that all species were pathogenic on wounded, green, and mature branches of the specified cultivars. Monthly inoculations of wounded, green, and lignified branches of Hass and Lamb Hass showed that both stem types were susceptible throughout the inoculation periods, regardless of the avocado phenological stage. In temperature-dependent growth and infection studies, growth of three points could vary during the growing season. Botryosphaeriaceae grown was higher between 20 and 30°C, but only Lasiodiplodia theobromae significantly grew and caused external lesions at 35°C. Lasiodiplodia theobromae also grew more on perseitol-amended media, all indicating its adaptation to warmer temperatures and capacity in metabolizing the avocado-produced sugar. Overall, this study extended our knowledge of the prevalence, identity, and pathogenicity of Botryosphaeriaceae on avocado cultivars, which will be useful to tailor management strategies.

Pistachio Male Inflorescences as an Alternative Substrate for the Application of Atoxigenic Strains of Aspergillus flavus.

Aflatoxins are carcinogens mainly produced by Aspergillus flavus and A. parasiticus in susceptible crops, including pistachio. The primary inoculum sources of these pathogens are plant debris in the orchard soils. In Californian fields, one approach to controlling aflatoxin contamination is based on releasing the atoxigenic strain of A. flavus AF36 in inoculated (coated) sorghum grains (AF36 Prevail). However, this control method can fail due to poor sporulation of the AF36 strain or sorghum grain losses due to predation. In 2008 and 2018, we showed that toxigenic and atoxigenic isolates of Aspergillus spp. frequently colonized fallen inflorescences of male pistachio trees. Under controlled conditions, strain AF36 profusely colonized pistachio male inflorescences when humidity was higher than 90%. However, there were significant differences between types of inflorescence (aerial > fallen). In 2016, we considerably (P = 0.015) increased the population of AF36 on the canopies of trees when fallen inflorescences were inoculated with AF36, compared with untreated trees. In 2017 and 2018, these differences were not detected (P > 0.05) due to cross-contamination of strain AF36 between seasons and neighboring plots. In any case, the density of AF36 spores on the canopy of the inflorescence-treated trees was similar (P > 0.05) to that on trees treated with the commercial product. Here, we present a new method for applying strain AF36 based on using a natural, abundant, and uniformly distributed substrate in pistachio fields, and we discuss how it can be improved. Furthermore, our results indicate that, in pistachio orchards where biocontrol practices are not conducted, eliminating this important source of toxigenic Aspergillus inoculum is recommended.

Frequency of Alternaria Genotypes Resistant to SDHI Fungicides in California Pistachio Orchards Determined by Real-Time PCR.

Real-time PCR methods were developed to quantify the frequency of SDHC-H134R and SDHB-H277Y mutants associated with succinate dehydrogenase inhibitors (SDHI) resistance in Alternaria populations from pistachio. The linearity of the standard curves demonstrated the applicability in the quantification of the assays. The accuracy and reliability of the qPCR protocols to determine the frequency of mutants in real samples were corroborated. Orchards visibly affected by Alternaria late blight were sampled. The frequency of mutants was determined using the qPCR assays, while the frequency of resistant phenotypes was determined using a single discriminatory dose. The statistical analysis showed that the frequencies of the mutation SDHC-H134R determined with the qPCR assay were highly correlated with those estimated with the conventional method. The survey also evidenced that resistance to boscalid is still widespread in California. Results also indicated the possible contribution of other mutations to SDHI resistance. Our results confirmed the prevalence of SDHC-H134R mutants and the occurrence of mutation SDHB-H277Y at low frequencies. The real-time PCR methods developed in this study were able to detect differences in the frequencies of resistant mutants caused by the use of chemical fungicides. Finally, the effects of two fungicide programs on the frequency of mutants resistant to SDHI and quinone outside inhibitors fungicides were studied using qPCR assays. The experiments demonstrated that the use of anilinopyrimidine and demethylation inhibitors fungicides in the same program reduced the frequency of these mutations in Alternaria populations. The qPCR methods developed and used in this study can be used to track resistance levels in the pistachio orchards on a large scale.

Effects of Cultivar Susceptibility, Branch Age, and Temperature on Infection by Botryosphaeriaceae and Diaporthe Fungi on English Walnut (Juglans regia).

Botryosphaeriaceae and Diaporthe fungi have been described as the main causal agents of branch dieback and shoot blight of English walnut (Juglans regia L.). To date, the effects of biotic and abiotic factors on disease development on this host are still poorly understood. Thus, the main goal of this study was to evaluate the effects of cultivar, shoot-branch age, and temperature on infection by Botryosphaeriaceae and Diaporthe fungi on English walnut. The susceptibility of eight commercial cultivars was evaluated against three Botryosphaeriaceae and two Diaporthe species. For the remaining experiments, shoots or branches of 'Chandler' were used. An initial experiment evaluating two inoculation methods was conducted, with inoculation with a mycelial plug being more consistent and useful than conidial suspension inoculation. Cultivar susceptibility varied depending on the fungal species, with 'Chandler' being among the most tolerant cultivars for shoot infection. One-year-old shoots were significantly more sensitive for both Neofusicoccum parvum and Diaporthe neotheicola in comparison with 2- to 4-year-old branches. The effect of temperature on shoot infection was evaluated under 5, 10, 15, 20, 25, 30, and 35°C. Lesion development was significantly higher for N. parvum isolates than for D. neotheicola isolates at all temperatures evaluated, with optimum temperature of shoot infection being ∼26°C for N. parvum and ∼21°C for D. neotheicola.

Characterization, pathogenicity, and fungicide sensitivity of Alternaria isolates associated with preharvest fruit drop in California citrus.

Alternaria rot has been recently described as an emerging fungal disease of citrus causing significant damage in California groves. A survey was conducted to determine latent infections on fruits, twigs, and leaves and investigate their seasonal patterns during 2019 and 2020. On fruits, latent infections were more associated with the stem end than with the stylar end, except during spring when a significantly high percentage of flowers (86%) had latent infections. Latent infections on twigs varied markedly between years (28% in 2019 and 9.5% in 2020), while Alternaria spp. were also recovered from citrus leaves. Alternaria isolates collected during the survey were identified based on multigene sequence analysis, confirming that Alternaria alternata and Alternaria arborescens are the two species associated with infections of citrus fruits. Of the 23 isolates, 19 were identified as A. alternata and demonstrated the dominance of this species over A. arborescens. Isolates representing populations of these two species were selected as representative isolates for physiological and morphological studies. A. alternata and A. arborescens showed similar conidial dimensions but differed in the number of conidia produced. Growth rates demonstrated that A. alternata grows faster than A. arborescens at all the temperatures evaluated, except at 25 and 35 °C. The growth patterns were similar for both species. The sporulation rate of the Alternaria isolates was influenced differently by temperature. This parameter also influenced conidial germination and appressorium formation, and no significant differences were observed between Alternaria species. Pathogenicity and aggressiveness tests on detached fruit demonstrated the ability of A. alternata and A. arborescens to cause internal lesions and produce fruit drop in the orchards with no quantitative differences between them (disease severity indexes of 58 and 68%, respectively). The fungicide sensitivity tests showed that DMI fungicides are the most effective fungicides in reducing mycelial growth. The SDHI fungicides had intermediate activity against the mycelial growth but also suppressed spore germination. The spore germination assay suggested that some of the isolates included in this study might have some level of resistance to QoI and SDHI fungicides. The findings of this study provide new information about the pathogens associated with the excessive fruit drop recently observed in some California citrus groves.

Characterization of Colletotrichum Isolates Causing Colletotrichum Dieback of Citrus in California.

Dieback caused by Colletotrichum spp. is an emerging disease in California citrus groves. A large-scale survey with emphasis on seasonal variations of latent infections was conducted throughout citrus orchards in Fresno, Kern, and Tulare counties in 2019 and 2020. Latent infections on citrus leaves and twigs varied markedly between years. Isolates of Colletotrichum spp. were obtained from asymptomatic tissue, and two groups were formed based on colony and spore morphology. The morphological groups were further identified based on multigene sequence analysis using the DNA regions ITS1-5.8S-ITS2, TUB2, and GAPDH. Results revealed that isolates belong to two phylogenetic species, C. gloeosporioides and C. karstii, being C. karstii more frequently isolated. Representative isolates of each species were further selected and characterized based on the response of physiological variables to temperature. Both species had similar optimum growth temperatures but differed in maximum growth rates, with C. gloeosporioides exhibiting a greater growth rate than that of C. karstii on media. Pathogenicity tests on citrus trees demonstrated the ability of C. gloeosporioides and C. karstii to cause lesions on twigs and no differences in aggressiveness. A fungicide screening performed in this study determined that the DMI fungicides were the most effective in reducing the mycelial growth of C. gloeosporioides and C. karstii. The QoI fungicides showed a remarkably inhibitory impact on spore germination of both species. On average, C. karstii was more sensitive to the DMI fungicides than C. gloeosporioides. The findings of this study provide new information to understand the Colletotrichum dieback of citrus.

Identification and characterization of Colletotrichum species associated with anthracnose on persimmon in Brazil.

Persimmon (Diospyros kaki) anthracnose is a major threat in production areas worldwide. Most of the studies are focused on Colletotrichum horii, but other species have been reported as well. The association of distinct Colletotrichum species present in Brazilian persimmon production regions as well as their host ranges are yet elusive. The aims of this work were to identify and characterize Colletotrichum species associated with the persimmon anthracnose. In a survey performed in four production regions of Brazil, 88.7% and 11.3% out of 231 isolates were identified as members of Colletotrichum gloeosporioides species complex (Cgc) or Colletotrichum acutatum species complex (Cac), respectively. A subset of 18 isolates were identified through multilocus phylogenetic analysis, using ITS-rDNA region and two loci, namely GAPDH and TUB2. This study revealed the presence of four species: C. horii (38.8%) and Colletotrichum fructicola (27.7%) from the Cgc and Colletotrichum nymphaeae (27.7%) and Colletotrichum melonis (5.8%), from the Cac. Additionally, 13 isolates were selected for morphological, physiological, and pathogenic analyses. Contrasting characteristics were observed among species of the Cgc and Cac complexes. The optimal temperature for mycelial growth and germination were higher for Cgc species. The percentage of appressoria melanisation also varied across complexes. All the identified species were able to cause anthracnose-like symptoms on persimmon fruit, leaves, shoots, and sepals. Colletotrichum species from persimmon were also able to infect apple and pear. The findings will support decisions to manage anthracnose of persimmon under high infection risk due to multiple host susceptibility.

Fungicide Resistance in Alternaria alternata from Blueberry in California and Its Impact on Control of Alternaria Rot.

Alternaria rot caused by Alternaria alternata is one of the major postharvest diseases affecting blueberries in California. The sensitivity profiles of A. alternata from blueberry field to quinone outside inhibitors (QoIs), boscalid, fluopyram, fludioxonil, cyprodinil, and polyoxin D in California were examined in this study. EC50 values of 51 A. alternata isolates for boscalid varied greatly among the isolates, ranging from 0.265 to >100 µg/ml. EC50 values of 51 A. alternata isolates to fluopyram, fludioxonil, cyprodinil, and polyoxin D were 5.188 ± 7.118, 0.078 ± 0.021, 0.465 ± 0.302, and 6.238 ± 7.352 µg/ml, respectively. In total, 143 isolates were screened for resistance at 5 and 10 µg/ml for fludioxonil, cyprodinil, and fluopyram, 10 µg/ml for polyoxin D, and 10 and 50 µg/ml for boscalid. Based on the published discriminatory concentrations for phenotyping resistance, of the 143 isolates, all were considered resistant to boscalid; 32, 69, and 42 were sensitive, low resistant, and resistant to fluopyram, respectively; and all were sensitive to fludioxonil and cyprodinil. In a PCR-restriction fragment length polymorphism method for phenotyping, 60 out of the 143 isolates were classified as resistant to QoIs. Control tests on detached blueberry fruit inoculated with different Alternaria isolates showed that fludioxonil and cyprodinil significantly reduced disease incidence and severity; however, pyraclostrobin, boscalid, fluopyram, and polyoxin D significantly reduced only disease severity. The obtained results will be helpful in making decisions on fungicide programs to control A. alternata isolates with resistance or reduced sensitivities to multiple fungicides.

Etiology of Botryosphaeria Panicle and Shoot Blight of Pistachio (Pistacia vera) Caused by Botryosphaeriaceae in Italy.

Pistachio (Pistacia vera) is an important crop in Italy, traditionally cultivated in Sicily (southern Italy) for several decades now. In recent years, new orchards have been planted in new areas of the island. Field surveys conducted in 2019 revealed the presence of symptomatic trees showing shoot dieback, cankers, fruit spots, and leaf lesions. Isolations from symptomatic samples consistently yielded fungal species in the Botryosphaeriaceae family. Identification of collected isolates was conducted using morphological and molecular analyses. Morphological characterization was based on conidia measurements of representative isolates and also effects of temperatures on mycelial growth was evaluated. DNA data derived from sequencing the ITS, tef1-α, and tub2 gene regions were analyzed via phylogenetic analyses (maximum parsimony and maximum likelihood). Results of the analyses confirmed the identity of Botryosphaeria dothidea, Neofusicoccum hellenicum, and N. mediterraneum. Pathogenicity tests were conducted on detached twigs and in the fields both on shoots as well as on fruit clusters using the mycelial plug technique. The inoculation experiments revealed that among the Botryosphaeriaceae species identified in this study N. hellenicum (occasionally detected) and N. mediterraneum were the most aggressive based on lesion length on shoots and fruits. N. mediterraneum was the most widespread among the orchards while B. dothidea can be considered a minor pathogen involved in this complex disease of pistachio. Moreover, to our knowledge, this is the first report of N. hellenicum in Italy.

Resistance to Aspergillus flavus and Aspergillus parasiticus in Almond Advanced Selections and Cultivars and Its Interaction with the Aflatoxin Biocontrol Strategy.

Aflatoxin contamination of almond kernels, caused by Aspergillus flavus and A. parasiticus, is a severe concern for growers because of its high toxicity. In California, the global leader of almond production, aflatoxin can be managed by applying the biological control strain AF36 of A. flavus and selecting resistant cultivars. Here, we classified the almond genotypes by K-Means cluster analysis into three groups (susceptible [S], moderately susceptible [MS], or resistant [R]) based on aflatoxin content of inoculated kernels. The protective effects of the shell and seedcoat in preventing aflatoxin contamination were also examined. The presence of intact shells reduced aflatoxin contamination >100-fold. The seedcoat provided a layer of protection but not complete protection. In kernel inoculation assays, none of the studied almond genotypes showed a total resistance to the pathogen. However, nine traditional cultivars and four advanced selections were classified as R. Because these advanced selections contained germplasm derived from peach, we compared the kernel resistance of three peach cultivars to that shown by kernels of an R (Sonora) and an S (Carmel) almond cultivar and five pistachio cultivars. Overall, peach kernels were significantly more resistant to the pathogen than almond kernels, which were more resistant than pistachio kernels. Finally, we studied the combined effect of the cultivar resistance and the biocontrol strain AF36 in limiting aflatoxin contamination. For this, we coinoculated almond kernels of R Sonora and S Carmel with AF36 72 h before or 48 h after inoculating with an aflatoxin-producing strain of A. flavus. The percentage of aflatoxin reduction by AF36 strain was greater in kernels of Carmel (98%) than in those of Sonora (83%). Cultivar resistance also affected the kernel colonization by the biological control strain. AF36 strain limited aflatoxin contamination in almond kernels even when applied 48 h after the aflatoxin-producing strain. Our results show that biocontrol combined with the use of cultivars with resistance to aflatoxin contamination can result in a more robust protection strategy than the use of either practice in isolation.

Update of pistachio leaf spot caused by Septoria pistaciarum in light of new taxonomic advances in Italy.

Septoria leaf spot is one the most widespread disease affecting pistachio (Pistacia vera) in countries of the Mediterranean region. Traditionally, three species have been associated with pistachio, including Septoria pistaciae, Septoria pistaciarum and S. pistacina. However, recent taxonomic studies have reordered and clarified the status of Septoria and septoria-like pathogens affecting pistachio. In our study, field surveys conducted in the traditional Sicilian pistachio production area of Bronte revealed the presence of trees showing characteristic septoria-like leaf spot. Collected isolates were morphologically and molecularly characterized. Morphological characterization was based on conidia measurements and evaluation of mycelial growth on different artificial media. Tested media included CMA, MEA, OA, PDA, and SNA. Phylogenetic analysis was conducted on a multi-locus approach (ITS + tef1 + tub2) based on Maximum Parsimony and Maximum Likelihood. Results showed that our isolates clustered with S. pistaciarum. Pathogenicity test was conducted in the field using conidia suspensions in order to fulfill Koch's postulates. Presence of characteristic rounded spots and pycnidia was evaluated on the inoculated leaves 9 and 23 days after inoculation. This study represents the first update on S. pistaciarum in Italy since its first identification in 1934.

Baseline Sensitivity of Alternaria alternata and A. arborescens to Natamycin and Control of Alternaria Rot on Stored Mandarin Fruit.

Alternaria rot caused by Alternaria alternata and A. arborescens is one of the major postharvest diseases on mandarin fruit in California. In this study, natamycin, a newly registered biofungicide, was evaluated for its potential as a postharvest treatment to control Alternaria rot on mandarin fruit. The baseline sensitivities of A. alternata and A. arborescens to natamycin were determined. Effective concentration inhibiting 50% of fungal growth (EC50) values of natamycin for 70 A. alternata isolates ranged from 0.694 to 1.275 µg/ml (mean = 0.921 µg/ml) in a conidial germination assay and from 2.001 to 3.788 µg/ml (mean = 2.797 µg/ml) for 40 A. alternata isolates in a mycelial growth assay. EC50 values of natamycin for 30 A. arborescens isolates ranged from 0.698 to 1.203 µg/ml (mean = 0.923 µg/ml) in a conidial germination assay and from 2.035 to 3.368 µg/ml (mean = 2.658 µg/ml) for 20 A. arborescens isolates in a mycelial growth assay. Control tests on detached mandarin fruit showed that natamycin at both low (460 µg/ml) and high (920 µg/ml) recommended rates significantly reduced disease incidence and severity on mandarin fruit inoculated with Alternaria isolates, regardless of species. High rate of natamycin significantly reduced disease incidence and severity compared with the nontreated control even when natamycin treatment was delayed for 6, 12, and 18 h after inoculation. Our results suggested that natamycin can be an effective postharvest fungicide for control of Alternaria rot on mandarin fruit.

Limited Evidence for Accumulation of Latent Infections of Canker-Causing Pathogens in Shoots of Stone Fruit and Nut Crops in California.

Prevalence of latent infections of the canker-causing fungi Botryosphaeria dothidea and species of Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum, and Phomopsis in young shoots of almond, prune, and walnut trees in California was studied to test the hypotheses that latent infections accumulate from current-season shoots to 1-year-old shoots in the orchard and there are distinct associations among pathogen taxa present as latent infections in the same shoot. Samples of newly emerged and 1-year-old shoots were periodically collected in each almond, prune, and walnut orchard for two growing seasons. A real-time quantitative PCR assay was used to quantify latent infection with three parameters: incidence, molecular severity, and latent infection index. Diplodia spp. were absent from most samples. For almond, Lasiodiplodia spp. and Cytospora spp. were detected with a maximum incidence >90%, while B. dothidea and Neofusicoccum spp. incidence was <20% in most cases. In prune orchards, the incidence levels of B. dothidea were >50% in most cases, while those of Cytospora spp. and Lasiodiplodia spp. were 30 to 60% and 30 to 100%, respectively. For walnut, many samplings showed higher incidence in 1-year-old (30 to 80%) than in newly emerged shoots (10 to 50%). Accumulation of latent infection between the two shoot age classes was detected in only a few cases. The percentages of samples showing coexistence of two, three, and four pathogen taxa in the same shoot were 20 to 25, <10, and <5%, respectively. Pairwise associations among pathogen taxa in the same shoot were significant in many cases.

Phylogenetic, Morphological, and Pathogenic Characterization of Alternaria Species Associated With Fruit Rot of Mandarin in California.

Alternaria rot caused by Alternaria species is one of the major postharvest diseases of mandarin fruit in California. The aims of this study were to identify these Alternaria species via phylogenetic analyses and morphological characteristics and test their pathogenicity on mandarin. Decayed mandarin fruits exhibiting Alternaria rot symptoms were collected from three citrus fruit packinghouses in the Central Valley of California. In total, 177 Alternaria isolates were obtained from decayed fruit and preliminarily separated into three groups representing three species (A. alternata, A. tenuissima, and A. arborescens) based on the colony characterization and sporulation patterns. To further identify these isolates, phylogenetic analysis was conducted based on DNA sequences of the second largest subunit of RNA polymerase II (RPB2), plasma membrane ATPase (ATPase), and Calmodulin gene regions in combination with morphological characters. Of the 177 isolates, 124 (70.1%) were identified as A. alternata, and 53 (29.9%) were A. arborescens. The isolates initially identified as A. tenuissima based on the morphological characteristics could not be separated from those of A. alternata in phylogenetic analysis and thus considered A. alternata. Pathogenicity tests showed that both Alternaria species were pathogenic on mandarin fruit at both 5°C and 20°C. Our results indicated that two Alternaria species, A. alternata and A. arborescens, were responsible for Alternaria rot of mandarin fruit in California, with A. arborescens causing fruit rot on mandarin being reported for the first time.

Quantification of the Aflatoxin Biocontrol Strain Aspergillus flavus AF36 in Soil and in Nuts and Leaves of Pistachio by Real-Time PCR.

The species Aspergillus flavus and A. parasiticus are commonly found in the soils of nut-growing areas in California. Several isolates can produce aflatoxins that occasionally contaminate nut kernels, conditioning their sale. Strain AF36 of A. flavus, which does not produce aflatoxins, is registered as a biocontrol agent for use in almond, pistachio, and fig crops in California. After application in orchards, AF36 displaces aflatoxin-producing Aspergillus spp. and thus reduces aflatoxin contamination. Vegetative compatibility assays (VCAs) have traditionally been used to track AF36 in soils and crops where it has been applied. However, VCAs are labor intensive and time consuming. Here, we developed a quantitative real-time PCR (qPCR) protocol to quantify proportions of AF36 accurately and efficiently in different substrates. Specific primers to target AF36 and toxigenic strains of A. flavus and A. parasiticus were designed based on the sequence of aflC, a gene essential for aflatoxin biosynthesis. Standard curves were generated to calculate proportions of AF36 based on threshold cycle values. Verification assays using pure DNA and conidial suspension mixtures demonstrated a significant relationship by regression analysis between known and qPCR-measured AF36 proportions in DNA (R2 = 0.974; P < 0.001) and conidia mixtures (R2 = 0.950; P < 0.001). Tests conducted by qPCR in pistachio leaves, nuts, and soil samples demonstrated the usefulness of the qPCR method to precisely quantify proportions of AF36 in diverse substrates, ensuring important time and cost savings. The outputs of this study will serve to design better aflatoxin management strategies for pistachio and other crops.

Further Investigation on Limb Dieback of Fig (Ficus carica) Caused by Neoscytalidium dimidiatum in California.

Fig limb dieback is a cosmopolitan disease caused by Neoscytalidium dimidiatum (Botryosphaeriaceae), characterized by branch and shoot cankers, discoloration of woody tissues, and dieback. The present study investigated the etiology of the disease in California that seems to have become prevalent among fig orchards in the last several years. During orchard surveys in Fresno, Kern, and Madera Counties over 3 years, we isolated consistently and evaluated the pathogenicity of N. dimidiatum under laboratory and field conditions. The effect of summer and winter pruning on the disease severity and the effects of different environmental and mechanical stresses, such as sunburn and wounding by mallets, were assayed. In addition, the susceptibility of six different cultivars and the effects of eradicating cankered shoots from the fig trees as a method to combat the spread of the disease were studied. Pathogenicity tests demonstrated that N. dimidiatum induces cankers on fig, mainly on wounded shoots. Results from the remaining experiments revealed that summer infection leads to more severe canker lesions than those induced by winter infection and that stressed shoots are more susceptible to infection than nonstressed shoots. 'Brown Turkey', 'Conadria', and 'Calimyrna' cultivars (all nonpersistent figs, i.e., needing pollination for fruit development) were less susceptible than the more susceptible 'Kadota', 'Sierra', and 'Black Mission' (all persistent figs, i.e., not needing pollination for fruit development). Canker removal from the orchard seems to be a good agronomic practice to avoid the spread of disease.