Influence of Planting Date and Cultivar on Diseases of Spring Durum Wheat.

In the semiarid regions of North Dakota and Montana, low annual precipitation favors production of high-quality durum wheat (Triticum turgidum subsp. durum). However, conducive weather conditions for disease epidemics have occurred more frequently in recent years. Modification of planting date can reduce disease risk by decreasing the timeframe in which a susceptible crop overlaps with conducive disease conditions. The effect of planting date on fungal leaf spotting diseases (leaf spot), ergot, Fusarium head blight (FHB), and yield of durum was evaluated in 11 experiments across four sites in eastern Montana and western North Dakota. Six durum cultivars with differing levels of susceptibility to leaf spot and FHB were planted at three planting dates from 2017 to 2019. Early planting maximized yield and influenced ergot incidence. Although there was no effect of planting date, reduced susceptibility to leaf spot and FHB was associated with a reduction in leaf spotting disease severity and deoxynivalenol, respectively, in the harvested grain. Growers in the semiarid regions of these states should prioritize the selection of disease-resistant cultivars to help manage sporadic disease outbreaks and continue to plant early to maximize yield.

Analysis of a putative glycosylation site in the Trichoderma virens elicitor SM1 reveals no role in protein dimerization.

The biocontrol fungus Trichoderma virens is an avirulent symbiont with the ability to control plant disease through the production of antibiotic compounds, induction of plant resistance to pathogens, and mycoparasitism of other fungi. Previous research has shown that resistance to plant pathogens in maize is induced by the secretion of a member of the cerato-platanin family of proteins, sm1, and that only the monomer of this protein has this activity. It has been hypothesized that glycosylation of sm1 disrupts dimer formation and keeps sm1 in this active monomer form. To further understand the role of this putative glycosylation site as a mechanism to prevent dimerization and subsequent elicitor activity, a point mutation was created in sm1 and transformed into a sm1 deletion strain. Analysis of the behavior of the altered protein (PTM) demonstrates that the putative glycosylation site is not involved in protein dimerization and deletion of this site does not prevent the protein from testing positive for glycosylation. We propose that SM1 is not glycosylated but instead may interact with an oligosaccharide or other small molecule. However, the exact mechanism of dimerization in SM1 remains unknown.

Effects on hyphal morphology and development by the putative copper radical oxidase glx1 in Trichoderma virens suggest a novel role as a cell wall associated enzyme.

Trichoderma spp. have been characterized for their capacity to act as biological control agents against several pathogens through the activity of secondary metabolites and cell wall degrading enzymes. However, only T. reesei has been widely studied for the ability to assimilate lignocellulose substrates. Protein analysis by SDS-PAGE of culture filtrate of T. virens revealed the presence of an unknown ∼77 kDa band protein (GLX1) that showed sequence homology to glyoxal-like oxidase genes involved in lignin degradation. The analysis and biochemical characterization of the 1,119 amino acid coded protein showed the presence of five carbohydrate-binding modules (CBMs) with affinity for colloidal chitin, and a functional glyoxal oxidase catalytic domain that is involved in the production of hydrogen peroxide when methylglyoxal was used as a substrate. The silencing of the glx1 gene resulted in mutants with more than 90% expression reduction and the absence of glyoxal oxidase catalytic activity. These mutants showed delayed hyphal growth, reduced colony and conidial hydrophobicity, but showed no changes in their biocontrol ability. Most significantly, mutants exhibited a loss of growth directionality resulting in a curled phenotype that was eliminated in the presence of exogenous H2O2. Here we present evidence that in T. virens, glx1 is not involved in the breakdown of lignin but instead is responsible for normal hyphal growth and morphology and likely does this through free radical production within the fungal cell wall. This is the first time that a glyoxal oxidase protein has been isolated and characterized in ascomycete fungi.

Microbial Resistance Mechanisms to the Antibiotic and Phytotoxin Fusaric Acid.

Fusaric acid (FA) produced by Fusarium oxysporum plays an important role in disease development in plants, including cotton. This non-specific toxin also has antibiotic effects on microorganisms. Thus, one expects a potential pool of diverse detoxification mechanisms of FA in nature. Bacteria and fungi from soils infested with Fusarium and from laboratory sources were evaluated for their ability to grow in the presence of FA and to alter the structure of FA into less toxic compounds. None of the bacterial strains were able to chemically modify FA. Highly FA-resistant strains were found only in Gram-negative bacteria, mainly in the genus of Pseudomonas. The FA resistance of the Gram-negative bacteria was positively correlated with the number of predicted genes for FA efflux pumps present in the genome. Phylogenetic analysis of predicted FA resistance proteins (FUSC, an inner membrane transporter component of the efflux pump) revealed that FUSC proteins having high sequence identities with the functionally characterized FA resistance protein FusC or Fdt might be the major contributors of FA resistance. In contrast, most fungi converted FA to less toxic compounds regardless of the level of FA resistance they exhibited. Five derivatives were detected, and the detoxification of FA involved either oxidative reactions on the butyl side chain or reductive reactions on the carboxylic acid group. The production of these metabolites from widely different phyla indicates that resistance to FA by altering its structure is highly conserved. A few FA resistant saprophytic or biocontrol strains of fungi were incapable of altering FA, indicating a possible involvement of efflux transporters. Deployment of both efflux and derivatization mechanisms may be a common feature of fungal FA resistance.

FUBT, a putative MFS transporter, promotes secretion of fusaric acid in the cotton pathogen Fusarium oxysporum f. sp. vasinfectum.

Fusaric acid (FA) is a key component in virulence and symptom development in cotton during infection by Fusarium oxysporum. A putative major facilitator superfamily (MFS) transporter gene was identified downstream of the polyketide synthase gene responsible for the biosynthesis of FA in a region previously believed to be unrelated to the known FA gene cluster. Disruption of the transporter gene, designated FUBT, resulted in loss of FA secretion, decrease in FA production and a decrease in resistance to high concentrations of FA. Uptake of exogenous FA was unaffected in the disruption transformants, suggesting that FA enters the cell in Fusarium by an independent mechanism. Thus, FUBT is involved both in the extracellular transport of FA and in resistance of F. oxysporum to this non-specific toxin. A potential secondary resistance mechanism, the production of FA derivatives, was observed in FUBT deletion mutants. Molecular analysis of key biochemical processes in the production of FA could lead to future host plant resistance to Fusarium pathogens.

Conversion of fusaric acid to Fusarinol by Aspergillus tubingensis: a detoxification reaction.

The fungus Fusarium oxysporum causes wilt diseases of plants and produces a potent phytotoxin fusaric acid (FA), which is also toxic to many microorganisms. An Aspergillus tubingensis strain with high tolerance to FA was isolated from soil and designated as CDRAt01. HPLC analysis of culture filtrates from A. tubingensis isolate CDRAt01 grown with the addition of FA indicated the formation of a metabolite over time that was associated with a decrease of FA. Spectral analysis and chemical synthesis confirmed the compound as 5-butyl-2-pyridinemethanol, referred to here as fusarinol. The phytotoxicity of fusarinol compared to FA was measured by comparing necrosis induced in cotton (Gossypium hirsutum L. cv. Coker 312) cotyledons. Fusarinol was significantly less phytotoxic than FA. Therefore, the A. tubingensis strain provides a novel detoxification mechanism against FA which may be utilized to control Fusarium wilt.

A putative terpene cyclase, vir4, is responsible for the biosynthesis of volatile terpene compounds in the biocontrol fungus Trichoderma virens.

A putative terpene cyclase vir4, which is a member of a secondary metabolite cluster, has been deleted in Trichoderma virens to determine its function. The deletion mutants were compared for volatile production with the wild-type as well as two other Trichoderma spp. This gene cluster was originally predicted to function in the synthesis of viridin and viridiol. However, the experimental evidence demonstrates that this gene cluster is involved in the synthesis of volatile terpene compounds. The entire vir4-containing gene cluster is absent in two other species of Trichoderma, T. atroviride and T. reesei. Neither of these two species synthesizes volatile terpenes associated with this cluster in T. virens. We have thus identified a novel class of volatile fungal sesquiterpenes as well as the gene cluster involved in their biosynthesis.

Functional characterization of a plant-like sucrose transporter from the beneficial fungus Trichoderma virens. Regulation of the symbiotic association with plants by sucrose metabolism inside the fungal cells.

• Sucrose exuded by plants into the rhizosphere is a crucial component for the symbiotic association between the beneficial fungus Trichoderma and plant roots. In this article we sought to identify and characterize the molecular basis of sucrose uptake into the fungal cells. • Several bioinformatics tools enabled us to identify a plant-like sucrose transporter in the genome of Trichoderma virens Gv29-8 (TvSut). Gene expression profiles in the fungal cells were analyzed by Northern blotting and quantitative real-time PCR (qRT-PCR). Biochemical and physiological studies were conducted on Gv29-8 and fungal strains impaired in the expression of TvSut. • TvSut exhibits biochemical properties similar to those described for sucrose symporters from plants. The null expression of tvsut caused a detrimental effect on fungal growth when sucrose was the sole source of carbon in the medium, and also affected the expression of genes involved in the symbiotic association. • Similar to plants, T. virens contains a highly specific sucrose/H(+) symporter that is induced in the early stages of root colonization. Our results suggest an active sucrose transference from the plant to the fungal cells during the beneficial associations. In addition, our expression experiments suggest the existence of a sucrose-dependent network in the fungal cells that regulates the symbiotic association.

Detoxification of Fusaric Acid by the Soil Microbe Mucor rouxii.

Fusarium oxysporum f. sp. vasinfectum race 4 (VCG0114), which causes root rot and wilt of cotton (Gossypium hirsutum and G. barbadense), has been identified recently for the first time in the western hemisphere in certain fields in the San Joaquin Valley of California. This pathotype produces copious quantities of the plant toxin fusaric acid (5-butyl-2-pyridinecarboxylic acid) compared to other isolates of F. oxysporum f. sp. vasinfectum (Fov) that are indigenous to the United States. Fusaric acid is toxic to cotton plants and may help the pathogen compete with other microbes in the soil. We found that a laboratory strain of the fungus Mucor rouxii converts fusaric acid into a newly identified compound, 8-hydroxyfusaric acid. The latter compound is significantly less phytotoxic to cotton than the parent compound. On the basis of bioassays of hydroxylated analogues of fusaric acid, hydroxylation of the butyl side chain of fusaric acid may affect a general detoxification of fusaric acid. Genes that control this hydroxylation may be useful in developing biocontrol agents to manage Fov.

A paralog of the proteinaceous elicitor SM1 is involved in colonization of maize roots by Trichoderma virens.

The biocontrol agent, Trichoderma virens, has the ability to protect plants from pathogens by eliciting plant defense responses, involvement in mycoparasitism, or secreting antagonistic secondary metabolites. SM1, an elicitor of induced systemic resistance (ISR), was found to have three paralogs within the T. virens genome. The paralog sm2 is highly expressed in the presence of plant roots. Gene deletion mutants of sm2 were generated and the mutants were found to overproduce SM1. The ability to elicit ISR in maize against Colletotrichum graminicola was not compromised for the mutants compared to that of wild type isolate. However, the deletion strains had a significantly lowered ability to colonize maize roots. This appears to be the first report on the involvement of an effector-like protein in colonization of roots by Trichoderma.