Characterization of Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina and Development of Diagnostic Tools for its Identification.

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

A maize lectin-like protein with antifungal activity against Aspergillus flavus.

The filamentous fungus Aspergillus flavus causes an ear rot on maize and produces a mycotoxin (aflatoxin) in colonized maize kernels. Aflatoxins are carcinogenic to humans and animals upon ingestion. Aflatoxin contamination results in a large loss of profits and marketable yields for farmers each year. Several research groups have worked to pinpoint sources of resistance to A. flavus and the resulting aflatoxin contamination in maize. Some maize genotypes exhibit greater resistance than others. A proteomics approach has recently been used to identify endogenous maize proteins that may be associated with resistance to the fungus. Research has been conducted on cloning, expression, and partial characterization of one such protein, which has a sequence similar to that of cold-regulated proteins. The expressed protein, ZmCORp, exhibited lectin-like hemagglutination activity against fungal conidia and sheep erythrocytes. Quantitative real-time PCR assays revealed that ZmCOR is expressed 50% more in maize kernels from the Mp420 line, a type of maize resistant to A. flavus, compared with the expression level of the gene in the susceptible B73 line. ZmCORp exhibited fungistatic activity when conidia from A. flavus were exposed to the protein at a final concentration of 18 mM. ZmCORp inhibited the germination of conidia by 80%. A 50% decrease in mycelial growth resulted when germinated conidia were incubated with the protein. The partial characterization of ZmCORp suggests that this protein may play an important role in enhancing kernel resistance to A. flavus infection and aflatoxin accumulation.

A maize trypsin inhibitor (ZmTIp) with limited activity against Aspergillus flavus.

Infection of maize both pre- and postharvest by Aspergillus flavus is a severe agricultural problem in the southern United States. Aflatoxins are secondary metabolites produced by A. flavus and are carcinogenic to humans and animals upon ingestion. Extensive research has been conducted to identify sources of resistance to A. flavus in maize. Some maize genotypes exhibit greater resistance to A. flavus than others. Many research groups have validated the role of plant trypsin inhibitors (TIs) as a means of plant defense against fungal infection. Research consisting of the cloning, expression, and partial characterization of one previously uncharacterized TI protein has been conducted. The overexpressed protein displayed TI activity, as expected, and some ability to alter germination of conidia (8%) from several fungal pathogens and to inhibit hyphal growth (30%). This effect on fungal growth, although less than that of previously investigated TIs, marks this protein as a potential source of resistance to aflatoxin accumulation in maize.

Inhibition of growth of Aspergillus flavus and fungal alpha-amylases by a lectin-like protein from Lablab purpureus.

Aspergillus flavus is a fungal pathogen of maize causing an important ear rot disease when plants are exposed to drought and heat stress. Associated with the disease is the production of aflatoxins, which are a series of structurally related mycotoxins known to be carcinogenic. Previous research has suggested that the alpha-amylase of A. flavus promotes aflatoxin production in the endosperm of infected maize kernels. We report here the isolation and characterization of a 36-kDa alpha-amylase inhibitor from Lablab purpureus (AILP). AILP inhibited the alpha-amylases from several fungi but had little effect on those from animal and plant sources. The protein inhibited conidial germination and hyphal growth of A. flavus. The amino acid sequence indicated that AILP is similar to lectin members of a lectin-arcelin-alpha-amylase inhibitor family described in common bean and shown to be a component of plant resistance to insect pests. AILP also agglutinated papain-treated red blood cells from human and rabbit. These data indicate that AILP represents a novel variant in the lectin-arcelin-alpha-amylase inhibitor family of proteins having lectin-like and alpha-amylase inhibitory activity.

Amy1, the alpha-Amylase Gene of Aspergillus flavus: Involvement in Aflatoxin Biosynthesis in Maize Kernels.

ABSTRACT Aspergillus flavus is the causal agent of an ear and kernel rot in maize. In this study, we characterized an alpha-amylase-deficient mutant and assessed its ability to infect and produce aflatoxin in wounded maize kernels. The alpha-amylase gene Amy1 was isolated from A. flavus, and its DNA sequence was determined to be nearly identical to Amy3 of A. oryzae. When Amy1 was disrupted in an aflatoxigenic strain of A. flavus, the mutant failed to produce extracellular alpha-amylase and grew 45% the rate of the wild-type strain on starch medium. The mutant produced aflatoxin in medium containing glucose but not in a medium containing starch. The alpha-amylase-deficient mutant produced aflatoxin in maize kernels with wounded embryos and occasionally produced aflatoxin only in embryos of kernels with wounded endosperm. The mutant strain failed to produce aflatoxin when inoculated onto degermed kernels. In contrast, the wild-type strain produced aflatoxin in both the endosperm and embryo. These results suggest that alpha-amylase facilitates aflatoxin production and growth of A. flavus from a wound in the endosperm to the embryo. A 14-kDa trypsin inhibitor associated with resistance to A. flavus and aflatoxin in maize also inhibited the alpha-amylase from A. flavus, indicating that it is a bifunctional inhibitor. The inhibitor may have a role in resistance, limiting the growth of the fungus in the endosperm tissue by inhibiting the degradation of starch.