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.

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.

Cross-Resistance Among Demethylation Inhibitor Fungicides With Brazilian Monilinia fructicola Isolates as a Foundation to Discuss Brown Rot Control in Stone Fruit.

Despite the resistance problems in Monilinia fructicola, demethylation inhibitor fungicides (DMIs) are still effective for the disease management of brown rot in commercial stone fruit orchards in Brazil. This study aims to investigate the sensitivity of M. fructicola isolates and efficiency of DMIs to reduce brown rot. A set of 93 isolates collected from Brazilian commercial orchards were tested for their sensitivities to tebuconazole, propiconazole, prothioconazole, and myclobutanil. The isolates were analyzed separately according to the presence or absence of the G461S mutation in MfCYP51 gene, determined by allele-specific test. The mean EC50 values for G461S mutants and wild-type isolates were respectively 8.443 and 1.13 µg/ml for myclobutanil, 0.236 and 0.026 µg/ml for propiconazole, 0.115 and 0.002 µg/ml for prothioconazole, and 1.482 and 0.096 µg/ml for tebuconazole. The density distribution curves of DMI sensitivity for both genotypes showed that myclobutanil and prothioconazole curves were mostly shifted toward resistance and sensitivity, respectively. Incomplete cross-resistance was detected among propiconazole and tebuconazole in both wild-type (r = 0.45) and G461S (r = 0.38) populations. No cross-sensitivity was observed among wild-type isolates to prothioconazole and the others DMIs tested. Fungicide treatments on detached fruit inoculated with M. fructicola genotypes showed significant DMI efficacy differences when fruit were inoculated with wild-type and G461S isolates. Protective applications with prothioconazole were more effective for control of both G461S and wild-type isolates compared with tebuconazole. Curative applications with tebuconazole were most effective in reducing the incidence and lesion size of G461S isolates. Sporulation occurred only for G461S isolates treated with tebuconazole under curative and preventative treatments. The differences found among the performance of triazoles against M. fructicola isolates will form the basis for recommendations of rational DMI usage to control brown rot in Brazil.

Understanding the Process of Latent Infection of Canker-Causing Pathogens in Stone Fruit and Nut Crops in California.

The Botryosphaeriaceae family is considered a fungal family that includes pathogens causing latent infection of woody plants, and a number of species were identified as causal pathogens of canker and shoot blight diseases. To better understand the process of latent infection of major canker-causing pathogens in woody tissues in different tree crops important in California, shoot and bud samples were randomly collected from four tree crops: almond, dried plum, pistachio, and walnut. The previously developed DNA primers and quantitative real-time PCR (qPCR) assay systems were applied to detect six canker-causing pathogen groups, including Botryosphaeria dothidea, and species of Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum, and Phomopsis. The concepts of molecular severity (MS) and latent infection index (LII) were introduced and applied to quantify the latent infection levels for these samples. Variation in incidence of latent infection among pathogen groups was observed, whereas the incidences were relatively low among species of Phomopsis and Diplodia. High incidences of Cytospora spp. were observed in two dried plum (prune) orchards. Most orchards showed high incidences of B. dothidea and Lasiodiplodia spp. and moderate incidences of Neofusicoccum spp. Variations in MS were observed among samples of the studied orchards, ranging from 4 to 8. The overall results of LII demonstrated that species of Diplodia and Phomopsis were less important in population development of canker-causing pathogens at the latent phase. Lasiodiplodia spp. were the most aggressive and had been well developed in populations among the studied tree crops. Cytospora spp. became predominant in two of the three dried plum orchards, whereas B. dothidea and Neofusicoccum spp. showed trends of increase in incidence across various tree crops. This study also demonstrated the usefulness of this sensitive qPCR approach in providing evidence of the latent phase of major canker-causing pathogens of stone fruit and nut crops at an early stage of latent infection in woody plant tissues.

The Point Mutation G461S in the MfCYP51 Gene is Associated with Tebuconazole Resistance in Monilinia fructicola Populations in Brazil.

The ascomycete Monilinia fructicola is the causal agent of brown rot of stone fruit in Brazil, causing major pre- and postharvest losses. For many years, the demethylation inhibitor (DMI) fungicide tebuconazole has been used as the most effective active ingredient for controlling brown rot and, as a result, strains of M. fructicola resistant to this ingredient have emerged in many Brazilian states producing stone fruit. The aim of this study was to investigate the mechanisms associated with the resistance of M. fructicola to DMI tebuconazole. By sequencing the M. fructicola CYP51 (MfCYP51) gene, encoding the azole target sterol 14α-demethylase, a mutation was identified at the nucleotide position 1,492, causing the amino acid substitution from glycine to serine at the codon position 461, associated with reduced tebuconazole sensitivity. In addition, it was observed that MfCYP51 gene expression could play a secondary role in DMI fungicide resistance of M. fructicola strains in Brazil. However, for the specific isolate found to exhibit elevated expression levels of MfCYP51, no insertions that would trigger gene expression were found. Based on the point mutation associated with tebuconazole resistance, an allele-specific polymerase chain reaction method was developed to quickly identify resistant genotypes within the Brazilian population. This is the first report determining molecular mechanisms for DMI resistance identification for M. fructicola isolates from Brazil. This information provides an important advancement for risk assessment of DMI fungicides used to manage brown rot of stone fruit.

Community Structure of Aspergillus flavus and A. parasiticus in Major Almond-Producing Areas of California, United States.

Several nut crops, including almond, pistachio, and walnut, can become contaminated with mycotoxins. Of greatest economic significance are aflatoxins, which are mainly produced by members of Aspergillus section Flavi. The distribution of the two sclerotial-size morphotypes of Aspergillus flavus (i.e., S and L strains) and A. parasiticus, the main species responsible for aflatoxin production among section Flavi, was monitored in the soil of almond orchards in California over a 5-year period from 2007 to 2011, excluding 2009. In total, 4,349 Aspergillus isolates were collected from 28 almond orchards located in the northern, central, and southern Central Valley in California. Overall, A. flavus L strain was the most frequent, followed by A. parasiticus and A. flavus S strain. However, variations in the spatial distribution of these three taxa were found between the three regions. Over the 5-year period, higher frequencies of L strain were more often observed in the southern region (79.9 to 95.1%, depending on year) compared with the northern region (21.4 to 47.1%). In the north, A. parasiticus was the most common strain, with frequencies of 28.5 to 61% for the various years. In addition, the frequency of aflatoxin-producing isolates among L strains fluctuated from year to year. A significant increase (P = 0.0001) was observed from 2008 (75% of the isolates produced aflatoxins) to 2007 (59%), and a decrease was observed from 2010 (61%) to 2011 (53%). Aflatoxin-producing L strain isolates were significantly more prevalent than atoxigenic isolates in each region during the 5-year survey, except in 2011 in the north, where more isolates were atoxigenic (56%) than aflatoxin-producing (44%). Our results indicate that the structure of A. flavus and A. parasiticus communities in the soil and the proportion of toxigenic isolates vary across regions and years. Such knowledge may help devise appropriate aflatoxin control strategies, including the use of atoxigenic isolates as biological control agents adapted to the soil environments in each region.