Zhonghua Ma

Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

ABSTRACT Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of theβ -tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.

Overexpression of the 14α-Demethylase Target Gene (CYP51) Mediates Fungicide Resistance in Blumeriella jaapii

ABSTRACT Sterol demethylation inhibitor (DMI) fungicides are widely used to control fungi pathogenic to humans and plants. Resistance to DMIs is mediated either through alterations in the structure of the target enzyme CYP51 (encoding 14α-demethylase), through increased expression of the CYP51 gene, or through increased expression of efflux pumps. We found that CYP51 expression in DMI-resistant (DMIR) isolates of the cherry leaf spot pathogen Blumeriella jaapii was increased 5- to 12-fold compared to that in DMI-sensitive (DMIS) isolates. Analysis of sequences upstream of CYP51 in 59 DMIR isolates revealed that various forms of a truncated non-long terminal direct repeat long interspersed nuclear element retrotransposon were present in all instances. Similar inserts upstream of CYP51 were not present in any of 22 DMIS isolates examined.

Biological control of aflatoxin contamination of crops

Aflatoxins produced primarily by two closely related fungi, Aspergillus flavus and Aspergillus parasiticus, are mutagenic and carcinogenic in animals and humans. Of many approaches investigated to manage aflatoxin contamination, biological control method has shown great promise. Numerous organisms, including bacteria, yeasts and nontoxigenic fungal strains of A. flavus and A. parasiticus, have been tested for their ability in controlling aflatoxin contamination. Great successes in reducing aflatoxin contamination have been achieved by application of nontoxigenic strains of A. flavus and A. parasiticus in fields of cotton, peanut, maize and pistachio. The nontoxigenic strains applied to soil occupy the same niches as the natural occurring toxigenic strains. They, therefore, are capable of competing and displacing toxigenic strains. In this paper, we review recent development in biological control of aflatoxin contamination.

Genetic Structure of Botrytis cinerea Populations from Different Host Plants in California

The population structure of Botrytis cinerea was investigated by using transposable elements, DNA fingerprinting generated by microsatellite primed-polymerase chain reaction (MP-PCR), and sensitivity to the hydroxyanilide fungicide, fenhexamid, for 234 isolates collected from fig, grape, kiwifruit, pea, and squash in California. Among 234 isolates tested, 195 had two transposable elements, Boty and Flipper (transposa type), 38 had only the Boty element (Boty type), and one had neither of these elements (vacuma type). Four of these 234 isolates, which belonged to the Boty type, were resistant to fenhexamid. A phenogram generated based on MP-PCR markers showed that the isolates were not clustered based on their source hosts or the presence of transposable elements. Analysis of molecular variance (AMOVA) showed that there were no significant genetic differentiations among isolates collected from grape, kiwifruit, pea, and squash at the Kearney Agricultural Center. A more detailed analysis based on AMOVA partition of the total genetic variance indicated that 96% of the variation occurred within populations. The parsimony tree length permutation (PTLPT) and index of association ( IA) analyses of MP-PCR data set were consistent with absence of sexual recombination in sampled populations of this pathogen.

Involvement of a Velvet Protein FgVeA in the Regulation of Asexual Development, Lipid and Secondary Metabolisms and Virulence in Fusarium graminearum

The velvet protein, VeA, is involved in the regulation of diverse cellular processes. In this study, we explored functions of FgVeA in the wheat head blight pathogen, Fusarium graminearum,using a gene replacement strategy. The FgVEA deletion mutant exhibited a reduction in aerial hyphae formation, hydrophobicity, and deoxynivalenol (DON) biosynthesis. Deletion of FgVEA gene led to an increase in conidial production, but a delay in conidial germination. Pathogencity assays showed that the mutant was impaired in virulence on flowering wheat head. Sensitivity tests to various stresses exhibited that the FgVEA deletion mutant showed increased resistance to osmotic stress and cell wall-damaging agents, but increased sensitivity to iprodione and fludioxonil fungicides. Ultrastructural and histochemical analyses revealed that conidia of FgVeA deletion mutant contained an unusually high number of large lipid droplets, which is in agreement with the observation that the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Serial analysis of gene expression (SAGE) in the FgVEA mutant confirmed that FgVeA was involved in various cellular processes. Additionally, six proteins interacting with FgVeA were identified by yeast two hybrid assays in current study. These results indicate that FgVeA plays a critical role in a variety of cellular processes in F. graminearum.

Characterization of Sterol Demethylation Inhibitor-Resistant Isolates of Fusarium asiaticum and F. graminearum Collected from Wheat in China

Fusarium asiaticum and F. graminearum are the primary causal agents of Fusarium head blight (FHB) of wheat in China. In this study, sensitivities of 159 F. asiaticum and F. graminearum isolates to a benzimidazole fungicide carbendazim (MBC) and to sterol demethylation inhibitors (DMIs) tebuconazole and prochloraz were determined. Among the 159 isolates, 9 were resistant to MBC and designated as MBC-R isolates. Three showed resistance to tebuconazole and prochloraz and designated as DMI-R isolates. There was no cross-resistance between MBC and DMI. Genetic analysis by microsatellite-primed polymerase chain reaction (PCR) showed that MBC-R or DMI-R isolates had different genotypes, which indicated that they originated from different wild-type parents. Analysis of two 14alpha-demethylase (cyp51) homologous genes (cyp51A and cyp51B) showed that the F. asiaticum isolates could be distinguished from F. graminearum isolates based on the sequence of cyp51A. Analysis of deduced amino acid sequence of cyp51A and cyp51B suggested that no mutations were associated with DMI resistance. Real-time PCR analysis showed that the DMI resistance was not related to the expression of cyp51A and cyp51B in F. asiaticum and F. graminearum, but expressions of both genes were induced greatly by the tebuconazole. Results of this study indicated that cyp51A would be an informative marker for analysis of population structure of F. asiaticum and F. graminearum, and the existence of homologous cyp51 genes in F. asiaticum and F. graminearum could provide new insights into DMI resistance in phytopathogenic fungi.

Effects of Water Stress on Botryosphaeria Blight of Pistachio Caused by Botryosphaeria dothidea

The effects of water potential (Ψ) on spore germination, germ tube elongation, mycelial growth of Botryosphaeria dothidea, and development of Botryosphaeria blight of pistachio caused by this fungus were studied in the laboratory and greenhouse. In laboratory tests, spore germination, germ tube elongation, and mycelial growth of B. dothidea increased as Ψ decreased from 0 to -2.0 MPa and declined as Ψ decreased below -2.0 MPa. Water stress (Ψ < -0.260 MPa) increased the severity of Botryosphaeria blight on young detached pistachio leaves. In greenhouse inoculation experiments, drought-stressed 2-year-old pistachio trees (leaf Ψ < -0.635 MPa) developed more severe Botryosphaeria blight disease than the nonstressed trees (Ψ > -0.485 MPa) in both 1999 and 2000 experiments. In a similar experiment using 1-year-old potted pistachio trees in 2000, withholding water treatments did not cause enough drought stress in pistachio leaves (Ψ > -0.584 MPa) and did not result in higher disease than the nonstressed trees (Ψ = -0.466 MPa). The correlation coefficient between Ψ and disease index for 1999 experiments was 0.954, and for 2000 experiments I and II was 0.981 and 0.939, respectively. These results indicate that drought stress can be a major predisposing factor of pistachio to infection by B. dothidea. Results from this study can be used to better understand the disease outbreaks in California pistachio orchards and to develop integrated management strategies by adjusting irrigation.

Quantification of airborne spores of Monilinia fructicola in stone fruit orchards of California using real-time PCR

The fungal pathogen Monilinia fructicola causes blossom blight and fruit brown rot of stone fruits in California. In this study, spore densities in the air were monitored in six orchard/year combinations with Burkard spore traps. A real-time PCR assay was developed to efficiently quantify the dynamics of spore density in these orchards during the growing season. Different patterns of dynamics of spore density were observed in these orchards. A linear relationship between numbers of spores counted with a compound microscope and those determined with the real-time PCR assay was obtained, using the same samples of spore traps. Spore density in five of six orchard/year combinations ranged from 0.0 to 0.05 spores l−1, except for that in orchard 4, which showed much higher values of spore density in the air, as well as higher values and wider range of incidences of blossom infection and fruit rot than those in the other orchards. The results demonstrated a potential method to quantitatively determine spore inoculum potential in orchards by using a real-time PCR assay.

Population Structure of Botryosphaeria dothidea from Pistachio and Other Hosts in California

ABSTRACT Genetic diversity was investigated among California populations of Botryosphaeria dothidea, causal agent of Botryosphaeria panicle and shoot blight of pistachio, with random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction (MP-PCR). We surveyed 120 isolates, 112 of which originated from the California Central Valley and included pistachio isolates (n = 52) and isolates from other plant species (n = 60). Out-group isolates (n = 8) were obtained from pistachio in Greece. There was a strong correlation (r = 0.99; P < 0.0001) between the RAPD- and MP-PCR dissimilarity data sets. Little genetic variation (haplotypic diversity [Hs] < 0.002) was detected among B. dothidea isolates collected from central and southern California pistachio plantings. We observed relatively high diversity for isolates from a northern California pistachio orchard (Hs = 0.0146), where the disease was first diagnosed, and from the Chico U.S. Department of Agriculture Germ Plasm Repository (Hs = 0.0726), where the first pistachio trees were planted in California in 1929. Isolates obtained from other hosts, especially those associated with the rare occurrence of the sexual stage of this fungus, showed the highest levels of diversity (Hs = 0.1689). Thirty-eight pistachio isolates (73.1%) had DNA fingerprints identical to 28 pycnidiospore-derived isolates (56.0%) obtained from other host species. Greenhouse inoculations demonstrated that all isolates obtained from other hosts were capable of infecting pistachio and produced characteristic disease symptomology. Thus, California populations of B. dothidea from pistachio are, for the most part, genetically uniform, with the sexual stage rare to absent. However, the rare occurrence of the sexual stage of B. dothidea on other hosts, and more importantly, the capacity of these isolates to infect pistachio, indicate that other host species may serve as sources of inoculum and genetic variation.

Paralogous cyp51 genes in Fusarium graminearum mediate differential sensitivity to sterol demethylation inhibitors

Analysis of the genome sequence of Fusarium graminearum revealed three paralogous cyp51 genes (designated cyp51A, -B, and -C) encoding 14-α demethylases in this fungus. Targeted gene disruption showed that the cyp51A, -B or -C disruption mutants were morphologically indistinguishable from the parent isolate on potato dextrose agar medium, which indicates that none of these genes is essential for mycelial growth. The sensitivity of cyp51A deletion mutants to seven sterol demethylation inhibitor (DMI) fungicides increased significantly compared to the parent strain, while sensitivity of cyp51C deletion mutants increased to some but not all DMIs. No change in DMI sensitivity was observed for cyp51B deletion mutants. The parental phenotypes of cyp51A and cyp51C deletion mutants were completely restored by genetic complementation with the wild-type cyp51A and cyp51C genes, respectively. The sensitivity of F. graminearum isolates increased significantly when subjected in vitro to a mixture of DMI fungicides triadimefon and tebuconazole as compared to the individual components. These results indicate that different DMI fungicides target different CYP51 proteins in F. graminearum and that a mixture of DMI fungicides can result in synergistic effects. Our findings have directly implications on chemical management strategies of plant diseases caused by Fusarium species.