Peanut Seed Coat Acts as a Physical and Biochemical Barrier against Aspergillus flavus Infection

Aflatoxin contamination is a global menace that adversely affects food crops and human health. Peanut seed coat is the outer layer protecting the cotyledon both at pre- and post-harvest stages from biotic and abiotic stresses. The aim of the present study is to investigate the role of seed coat against A. flavus infection. In-vitro seed colonization (IVSC) with and without seed coat showed that the seed coat acts as a physical barrier, and the developmental series of peanut seed coat showed the formation of a robust multilayered protective seed coat. Radial growth bioassay revealed that both insoluble and soluble seed coat extracts from 55-437 line (resistant) showed higher A. flavus inhibition compared to TMV-2 line (susceptible). Further analysis of seed coat biochemicals showed that hydroxycinnamic and hydroxybenzoic acid derivatives are the predominant phenolic compounds, and addition of these compounds to the media inhibited A. flavus growth. Gene expression analysis showed that genes involved in lignin monomer, proanthocyanidin, and flavonoid biosynthesis are highly abundant in 55-437 compared to TMV-2 seed coats. Overall, the present study showed that the seed coat acts as a physical and biochemical barrier against A. flavus infection and its potential use in mitigating the aflatoxin contamination.

Fusarium verticillioides induces maize-derived ethylene to promote virulence by engaging fungal G-protein signaling

Seed maceration and contamination with mycotoxin fumonisin inflicted by Fusarium verticillioides is major disease of concern for maize producers world-wide. Meta-analyses of QTL for Fusarium ear rot resistance uncovered several ethylene (ET) biosynthesis and signaling genes within them, implicating ET in maize interactions with F. verticillioides. We tested this hypothesis using maize knock-out mutants of the 1-aminocyclopropane-1-carboxylate (ACC) synthases, ZmACS2 and ZmACS6. Infected wild-type seed emitted five-fold higher ET levels compared to controls, whereas ET was abolished in the acs2 and acs6 single and double mutants. The mutants supported reduced fungal biomass, conidia and fumonisin content. Normal susceptibility was restored in the acs6 mutant with exogenous treatment of ET precursor, ACC. Subsequently, we showed that fungal G-protein signaling is required for virulence via induction of maize-produced ET. F. verticillioides Gβ subunit and two regulators of G-protein signaling mutants displayed reduced seed colonization and decreased ET levels. These defects were rescued by exogenous application of ACC. We concluded that pathogen-induced ET facilitates F. verticillioides colonization of seed, and in turn host ET production is manipulated via G-protein signaling of F. verticillioides to facilitate pathogenesis.

Single Marker Analysis in Groundnut for Tolerance to in vitro Seed Colonization by Aspergillus flavus and Aflatoxin Contamination

Background: Aflatoxin contamination in groundnut is a serious health concern for both humans and ruminants. Genetic resistance is a viable, cost-effective and eco-friendly approach to manage aflatoxin contamination. Molecular markers particularly SSRs have been proved to be very effective in Marker Assisted Selection.Methods: In this study, single marker analysis using 30 SSR markers in 66 groundnut genotypes was executed to know if any selected SSRs were linked to in vitro seed colonization by Aspergillus flavus (IVSCAF) and/or aflatoxin contamination.Result: Single marker analysis revealed significant association of few SSR markers with tolerance to IVSCAF and/or aflatoxin contamination. Four markers viz., GM-1954, GM-1883, pPGPseq-2F05 and S-03 were found to be associated with in vitro seed colonization by A. flavus. The marker GM-1954 has shown a maximum R2 value of 14.07 indicating that 14.07 per cent phenotypic variation for IVSCAF has been explained by this marker (F=0.002**). Further, three markers viz., S-21, S-80 and GM-1954 were found to be associated with tolerance to aflatoxin contamination. It is evident from the results that, the marker GM 1954 has shown association with both IVSCAF as well as aflatoxin contamination. However, the R2 value of GM 1954, which explains the phenotypic variation for aflatoxin contamination, was less (6.21) as compared to that of IVSCAF (14.07).

Bacteria Detected in both Urine and Open Wounds in Nursing Home Residents: a Pilot Study

ABSTRACT Nursing home residents are at a greater risk of developing pressure injuries that develop into an open wound, which can become colonized with bacteria. Understanding the factors that influence microbial colonization of open wounds can lead to the prevention of infections. The relationship between bacteria found in urine and those in open wounds is currently unknown. To determine if bacterial species colonizing open wounds are also found in the urine, we conducted a pilot study with nursing home residents, comparing bacterial species present in the urine with those present in wounds between the umbilicus and mid-thigh. To identify microbial species that were present in both urine and open wound at one time point in one patient, standard clinical bacteriologic culture techniques followed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) were used, as well as 16S rRNA-encoding gene amplicon sequencing. We found some bacterial species detected in urine were also detected in open wounds in one individual at one time point, using both culture-dependent and -independent techniques. Bacterial species that were more often detected, using culture-dependent and -independent methods, at both sites included Enterococcus faecalis, Proteus mirabilis, Escherichia coli, and Providencia stuartii. This pilot study provides evidence that bacterial species identified within the urine can also be identified in open wounds in the same patient at one point in time. Further studies are needed to investigate if these species are of the same lineage and if the urinary microbiota are able to seed colonization of open wounds below the umbilicus. IMPORTANCE Older adults, specifically those in nursing facilities, are more susceptible to developing chronic open nonhealing wounds. Chronic open nonhealing wounds severely impact an individual’s quality of life and can lead to other comorbidities, such as infection. Recent evidence suggests that the open wound bacterial community can influence wound healing and repair. It is important to understand all sources of open wound contamination to improve preventative infection measures and treatment protocols. In this pilot study, we investigated if bacterial species isolated from urine can also be isolated from open wounds located between the levels of the umbilicus and mid-thigh in the same patient at the same point in time. A growing body of evidence suggests that urine can harbor a microbial community, even in asymptomatic individuals, and older adults are more prone to urinary incontinence. This is the first study to investigate bacterial species concordance between these two anatomical sites. We found, using both culture-dependent and -independent methods, that the same bacterial species can colonize both the urine and wound in one patient at one point in time. Further studies are needed to investigate if these species are of the same lineage and if the urinary microbiota are able to seed colonization of these types of open wounds.

Distinct Roles for VeA and LaeA in Development and Pathogenesis of Aspergillus flavus

ABSTRACT Aspergillus flavus, a mycotoxigenic filamentous fungus, colonizes several important agricultural crops, such as maize and peanuts. Two proteins, VeA and LaeA, known to form a nuclear complex in Aspergillus nidulans have been found to positively regulate developmental processes in several Aspergillus species. Here, an examination of near-isogenic A. flavus mutants differing in copy number of veA and laeA alleles (0, 1, or at least 2 each) revealed critical roles for VeA and LaeA in A. flavus development and seed colonization. In contrast to the wild type, both null mutants were unable to metabolize host cell lipid reserves and were inhibited by oleic acid in growth assays. The copy number of LaeA but not VeA appeared critical for a density-dependent sclerotial-to-conidial shift, since the multicopy laeA (MClaeA) strain produced relatively constant sclerotial numbers with increasing population size rather than showing the decrease in sclerotia seen in both the wild-type and MCveA strains. The MCveA-laeA strain yielded an intermediate phenotype. This study revealed unique roles of VeA and LaeA in seed pathogenesis and fungal biology, distinct from their cooperative regulatory functions in aflatoxin and sclerotial development.

Relationships Between Oleic and Linoleic Acid Content and Seed Colonization by Cercospora kikuchii and Diaporthe phaseolorum

Compared with standard cultivars, seed of mid-oleic soybean genotypes sometimes have shown increased colonization by Cercospora kikuchii in the field as judged by increased levels of purple-stained seed. To examine relationships between oleic and linoleic acid levels in soybean seed and postharvest seed colonization by two fungal seed pathogens, we inoculated seed with differing oleic:linoleic acid (O/L) ratios. Seed with defined O/L ratios were produced by allowing seed development of two isogenic soybean lines to occur in three different air temperature environments. Seed produced in these environments were harvested, individually analyzed for fatty acid composition, and inoculated with mycelium preparations of the fungal seed pathogens C. kikuchii or Diaporthe phaseolorum var. sojae. Fungal biomass of infected seed was quantified by measuring in vitro ergosterol content. For both soybean lines, colonization by C. kikuchii was positively correlated with the O/L ratio (r = 0.55, P < 0.03) and oleic acid content (r = 0.61, P < 0.02), and negatively correlated with linoleic (r = –0.60, P < 0.02) and linolenic (r = –0.58, P < 0.03) acid content. No association was found between the extent of seed colonization by D. phaseolorum and the seed O/L ratio. Our data suggest that the O/L ratio may be related to soybean seed colonization by C. kikuchii, but there is no evidence of a relationship with D. phaseolorum var. sojae colonization.

Differential Interference with Pythium ultimum Sporangial Activation and Germination by Enterobacter cloacae in the Corn and Cucumber Spermospheres

ABSTRACT Differential protection of plants by Enterobacter cloacae was studied by investigating early sensing and response behavior of Pythium ultimum sporangia toward seeds in the presence or absence of E. cloacae. Ten percent of P. ultimum sporangia were activated within the first 30 min of exposure to cucumber seeds. In contrast, 44% of the sporangia were activated as early as 15 min after exposure to corn seeds with over 80% sporangial activation by 30 min. Germ tubes emerged from sporangia after 2.5 and 1.0 h in the cucumber and corn spermospheres, respectively. Seed application of the wild-type strain of E. cloacae (EcCT-501R3) reduced sporangial activation by 45% in the cucumber spermosphere, whereas no reduction was observed in the corn spermosphere. Fatty acid transport and degradation mutants of E. cloacae (strains EcL1 and Ec31, respectively) did not reduce sporangial activation in either of the spermospheres. Although wild-type or mutant strains of E. cloacae failed to reduce seed colonization incidence, pathogen biomass on cucumber seeds was reduced in the presence of E. cloacae strains EcCT-501R3 and Ec31 by 4 and 8 h after sowing, respectively. By 12 h, levels of P. ultimum on cucumber seeds treated with E. cloacae EcCT-501R3 did not differ from levels on noninoculated seeds. On corn seeds, P. ultimum biomass was not affected by the presence of any E. cloacae strain. When introduced after sporangial activation had occurred, E. cloacae failed to reduce P. ultimum biomass on cucumber seeds compared with that on nontreated seeds. Also, increasing numbers of sporangia used to inoculate seeds yielded increased pathogen biomass at each sampling time. This indicates a direct link between the level of seed-colonizing biomass of P. ultimum and the number of activated and germinated sporangia in the spermosphere, suggesting that E. cloacae suppresses P. ultimum seed infections by reducing sporangial activation and germination within the first 30 to 90 min after sowing.