Immunohistochemical investigation of cotton carpel tissue exposed to xylanolytic hydrolases of Aspergillus flavus

Cotton carpel tissue (35–45 days post-anthesis) that had been treated with a mixture of xylanolytic hydrolases derived from Aspergillus flavus was subjected to immunocytochemical analysis. Microscopic examination of treated tissues revealed severe degradation of the secondary wall structure. Control tissue cells revealed the presence of high concentrations of xylans/arabinoxylans throughout the cell wall, as well as significant concentrations of arabinogalactan proteins in secondary wall structure. Carpel cells treated with a mixture of A. flavus-produced xylanolytic hydrolases showed a much reduced presence of labeling by xylan-specific antibodies on the inner wall surface, suggesting a severe loss of these plant polysaccharides in the secondary wall structure. Carpel exposure to a purified 14 kD endoxylanase from A. flavus also resulted in a severe reduction of xylans from secondary wall structure, although penetration of the tissue was not as dramatic. Arabinogalactan proteins were not as severely affected by the xylanolytic hydrolases. Comparison of control tissue with hydrolase-treated tissue stained with toluidine blue revealed an apparent reduction in wall thickness, supporting the conclusion of secondary wall structure degradation. Interestingly, the pectins could only be detected in the samples treated with xylanolytic enzymes, indicating that the pectins were being masked by xylans. These results are consistent with the conclusion that the xylanolytic hydrolase complex of A. flavus is a critical factor for host cell wall maceration and may represent another important fungal virulence factor, in addition to pectolytic hydrolase activities.     

Influence of plant host species on intraspecific competition during infection by Aspergillus flavus

Compositions of Aspergillus flavus populations determine the extent to which crops become contaminated with aflatoxins. In the current study, influences of diverse crop hosts on competition among A. flavus isolates were quantified with pyrosequencing. Maize, cotton, soyabean and sorghum supported different levels of sporulation, but intraspecific differences in sporulation were not detected on any host. However, hosts differentially influenced competition during infection, allowing greater sporulation by some isolates and increased host tissue invasion by others. Furthermore, competitive interactions during host invasion did not predict isolate success during sporulation. Isolates were similarly competitive on maize and sorghum, the two most closely related hosts. Host‐specific influences on intraspecific competition may dictate compositions of A. flavus populations and, as a result, the severity of aflatoxin contamination. Host factors should be considered when designing and implementing aflatoxin management strategies including biocontrol with atoxigenic strains.     

Aspergillus flavus-secreted proteins during an in vivo fungal-cotton carpel tissue interaction

Cotton bolls (Gossypium hirsutum) were inoculated with Aspergillus flavus to investigate extracellular hydrolases produced during infection. Fungal proteins from the infection site were isolated and analyzed by SDS-PAGE. Protein bands were excised and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified several proteins including an exoglucanase 1 precursor (AFL2G_03805), a hypothetical protein similar to rhamnogalacturonan lyase A (AFL2G_05136), and a hypothetical protein similar to pectate lyase A (AFL2G_05954). We also matched three peptides to an oryzin precursor protein (AFL2G_01995). Our findings support the conclusion that glucanase, pectinolytic, and proteolytic proteins are important for fungal maceration of cotton carpel tissue during infection.     

Potential involvement of Aspergillus flavus laccases in peanut invasion at low water potential

Aspergillus flavus accumulates carcinogenic aflatoxins in peanuts, mainly in immature kernels during drought. Aspergillus flavus invasion induces accumulation of phytoalexins, mostly stilbenoids in peanut, as a plant defence mechanism. Because fungal laccases are often related to pathogenicity and can degrade stilbenoids, this study reports for the first time the expression of A. flavus laccases in the presence of kernels, hulls and low water potential in relation to the accumulation of phytoalexins in peanut kernels. Packed‐cell volume (PCV) of A. flavus biomass was significantly higher (P ≤ 0·01) in the presence of mature kernels, dead kernels, and mature and immature peanut hulls than the control. The presence of kernels and hulls lowered the level of expression of three A. flavus laccases by 4–6‐fold (P < 0·01), whereas 3% sucrose up‐regulated them by 35–304‐fold, and low water potential (?1·1 MPa) up‐regulated them by 85–248‐fold (P < 0·01). Phytoalexins that accumulated in peanut kernels in the presence of A. flavus and were quantified by HPLC‐DAD‐MS were primarily the stilbenoids: 3′‐isopentadienyl‐3,5,4′‐trihydroxystilbene (IPD), chiricanine‐A, arachidin‐2, arachidin‐3 and arahypin‐1. Apparent degradation of phytoalexins was observed when using a priori induction of phytoalexins in seeds in combination with a priori induction of laccases in A. flavus. The up‐regulation of laccase expression observed at ?1·1 MPa and at high sucrose concentration could be contributing to peanut invasion in immature kernels under drought conditions.     

Comparison of different inoculating methods to evaluate the pathogenicity and virulence of Aspergillus niger on two maize hybrids

A two-year field study was conducted to determine the effects of artificial inoculation techniques on the pathogenicity and virulence of Aspergillus niger kernel infection on two maize hybrids. Test plants included in the study were hybrids resistant and susceptible to Aspergillus flavus to determine if the host resistance mechanisms that limited A. flavus infection would also suppress A. niger infection. Ears were inoculated with the silk-channel, side-needle, and spray techniques 7?days after midsilk (50% of the plants in a plot had silk emerging). Ears were also inoculated with a modified-pinbar technique 21?days after midsilk. Kernel infection in 2008 in inoculated plants ranged from 2% to 11% and from 2% to 45% in the resistant and susceptible hybrids, respectively. In 2009, kernel infection in inoculated plants ranged from 13% to 32% and from 10% to 67% in the resistant and susceptible, respectively. The silk-channel, side-needle, and modified-pinbar techniques produced significantly higher levels of kernel infection in the susceptible hybrid in both years than the spray technique. When hybrids were compared, the silk-channel, side-needle, and modified-pinbar techniques induced significantly higher levels of infections in the susceptible hybrid than in the resistant hybrid in 2008 and 2009. The level of A. niger pathogenicity and virulence increased when conidia were placed inside the husks of developing ears by wounding (modified-pinbar and side-needle techniques) or non-wounding (silk-channel technique) inoculation methods. Although A. niger kernel infection was significantly lower in the A. flavus resistant hybrid compared to the A. flavus susceptible hybrid, A. niger infection levels were much higher than A. flavus infection levels typically observed in both of these hybrids in past studies.     

Cloning and characterization of an NBS-LRR resistance gene from peanuts (Arachis hypogaea L.)

The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family accounts for the largest number of known disease resistance genes and is one of the largest gene families in plant genomes. In this study, resistance gene analogs (RGAs) were isolated from peanuts based on the NBS domain. A full-length cDNA, PnAG₃, was obtained by rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the length of PnAG₃ was 1882 bp, which included a complete open reading frame of 1335 bp that encoded for the PnAG₃ protein composed of 444 amino acids. Multiple analyses showed that this protein had homology with known resistance proteins, the highest being 48.01% with a resistance protein from Arachis cardenasii. The polypeptide has a typical non-TIR-NBS-LRR gene structure. Real-time fluorescence quantitative PCR analysis showed that after Aspergillus flavus infection, expression of the PnAG₃ gene in J11 (A. flavus-resistant species) increased by 16.68, 11.16 and 25.96 in the seed coat, kernel and pericarp, respectively. However, it only increased 2–3 times in JH1012 (A. flavus-sensitive species). Cloning of the putative resistance gene from peanut provides a basis for studying the structure and function of peanut disease resistance-related genes and disease resistance genetic breeding in peanuts.     

Distribution and genetic diversity among Aspergillus flavus isolates across three agro-ecologies essential for maize cultivation in Ghana

Fungal mycotoxins are important contaminants of agricultural commodities that pose serious concerns to producers, consumers and exporters. Aflatoxin is a carcinogenic metabolite produced by the fungi Aspergillus flavus and A. parasiticus. These fungi and other mould species living in the soil contaminate several crops including maize. This study was undertaken with the following objectives: (i) to assess the presence of A. flavus and other moulds in Ghanaian soil; (ii) to determine the distribution and density of A. flavus under three agro-ecologies noted for major maize production; and (iii) to assess the effect of percentage of carbon (C) and nitrogen (N) and soil pH on A. flavus population densities and diversity in the isolates found across the agro-ecologies. The data showed seven moulds that were common across the agro-ecologies. Significant differences (P < 0.05) were observed in the A. flavus density and distribution within and across these agro-ecologies. Fumesua soils recorded the highest levels of A. flavus (1.185 × 10³ cfu g⁻¹) while Akomadan recorded the least (9.76 × 10² cfu g⁻¹). Percentage available C, N and soil pH did not significantly influence A. flavus density. The A. flavus isolates identified in this study varied in genetic sequence within the aflatoxin gene cluster, but these differences were not distinguishable by origin.     

Factors associated with the incidence of Aspergillus section Flavi and aflatoxin contamination of peanuts in the Busia and Homa bay districts of western Kenya

The aim of this study was to identify agronomic, ecological and sociocultural factors that could be modified to reduce the risk of aflatoxin contamination of peanuts from western Kenya. Presence of fungi within section Flavi of the genus Aspergillus and levels of total aflatoxin were determined for 436 peanut samples from the Busia and Homa bay districts. A total of 1458 cultures of Aspergillus flavus or A. parasiticus isolated from the samples were assayed for production of aflatoxin B₁, B₂, G₁ and G₂. Associations among the incidences of fungal species, incidences of samples with ≥10 μg kg^?1 aflatoxin, production of specific aflatoxin types and various agronomic, ecological and sociocultural factors were modelled with chi‐squared and logistic regression methods. The predominant species were A. flavus L‐strain (78% incidence), A. flavus S‐strain (68%) and A. niger (65%). Occurrence of A. caelatus, A. alliaceus and A. tamarii in Kenya was also documented. Samples from the Busia district were three times (odds ratio = 3·01) as likely to contain ≥10 μg kg^?1 of total aflatoxin as were samples from the Homa bay district, while samples containing A. flavus S‐strain were 96% more likely to exceed this threshold compared with samples from which this fungus was not isolated. Grading, planting improved cultivars and membership of a producer marketing group were negatively associated with the incidence of A. flavus, while crop rotation was negatively correlated with the incidence of B aflatoxins. These sociocultural factors can be modified to reduce the risk of peanut contamination with aflatoxin.     

Functional characterization of a new pathogen induced xylanase inhibitor (RIXI) from rice

Rice xylanase inhibitor RIXI is a XIP-type inhibitor that belongs to the glycoside hydrolase family 18 (GH18), which includes plant class III chitinases (EC 3.2.1.14) known as PR-8 proteins. The aim of this study was to elucidate whether RIXI had any effect on rice endoxylanase and its role(s) in plant defence. RIXI encoding sequence was cloned from rice (Oryza sativa cv. Nipponbare), and was expressed in Escherichia coli. The activity of recombinant RIXI was investigated – among the tested xylanases, the GH11 xylanase from Aspergillus niger was the most inhibited, while rice endogenous xylanase OSX was least inhibited. Semi-quantitative real-time polymerase chain reaction (PCR) and quantitative real-time PCR showed that the xylanase inhibitor gene RIXI was up-regulated in rice seedlings infected by Magnaporthe grisea. The increased RIXI expression was also accompanied by significantly elevated expression of pathogenesis-related protein gene (PR-1) and class III chitinase gene (Cht-3). This suggested that RIXI might be involved in environmental responses such as defense against phytopathogens.     

Fungal derived cytokinins are necessary for normal Ustilago maydis infection of maize

Phytohormones derived from fungi play a key role in regulating plant–pathogen interactions; however, deciphering the separate contributions of pathogen and plant during infection has been difficult. Here, the Ustilago maydis–Zea mays pathosystem was used to investigate this chemical exchange. Ustilago maydis, the causative agent of maize smut, produces cytokinins (CK), which are a group of phytohormones responsible for directing plant development. The characteristic symptom of smut disease is the formation of tumours composed of plant and fungal tissue. Isopentenyltransferase (IPT) catalyses the rate‐limiting step in CK biosynthesis, and U. maydis strains in which the sole tRNA‐ipt gene was deleted no longer produced CKs. These deletion strains elicited fewer, smaller tumours than the pathogenic strain SG200. High performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI MS/MS) was used to detect and quantify phytohormone levels in infected tissue. This revealed that key hormone changes in SG200 infections were not present in infections by deletion strains, suggesting that CK production by U. maydis is required for the altered phytohormone profile in infected tissue relative to uninfected tissue. Separate analyses indicated that U. maydis tRNA‐ipt mutants might be altered in their ability to metabolize CKs taken up from the environment. Mining the U. maydis genome identified genes encoding putative CK signalling and biosynthesis proteins.     

Ss-Bi1 encodes a putative BAX inhibitor-1 protein that is required for full virulence of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a destructive necrotrophic plant pathogen with global distribution. Although S. sclerotiorum has been studied extensively, substantial research on aspects of the pathogen's ability to cause disease is still needed. Bax inhibitor-1 protein functions as a suppressor of programmed cell death and is involved in the response to biotic and abiotic stress in animals, plants and yeast. In this study, we functionally characterized a putative Bax inhibitor-1 protein, Ss-Bi1, from S. sclerotiorum. Ss-Bi1 is predicted to contain a BAX inhibitor-1-like super family domain and shows significant homology with many BAX inhibitor-1 proteins. High expression levels of Ss-Bi1 were observed in hyphae under various stresses. Targeted silencing of Ss-Bi1 resulted in reduced virulence in host plants. Ss-Bi1 gene-silenced strains were more sensitive to heat stress and ER stress than the wild-type strain. The results suggest that Ss-Bi1 encodes a putative BAX inhibitor-1 protein that is required for full virulence of S. sclerotiorum.     

Induction of defence responses against Erwinia soft rot by an endogenous pectate lyase in potatoes

Pectate lyase (PL) enzymes are major virulence factors of Erwinia carotovora (Ec) bacteria. They degrade plant cell wall pectin into unsaturated oligogalacturonates (OG) known to elicit plant defence responses. Therefore, a gene encoding the isoenzyme PL3 of Ec ssp. atroseptica was transformed by means of Agrobacterium into potatoes of cv. Désirée. Four PL-transgenic potato plant lines selected on the basis of greenhouse experiments were grown over a period of 4 years (1997–2000) in the field. It is shown that the heterologous PL enzyme mediated an enhanced resistance to Erwinia soft rot in field grown tubers. Thus compared to the non-transgenic counterpart extension of rotting was significantly reduced (P < 0.001) on the wound surface of PL-expressing tubers. Moreover, the threshold density ofEc -bacteria causing a progressive soft rot was up to 19-fold higher on tuber tissue containing the PL enzyme. An induction of plant defence responses in PL-transgenic potatoes may be indicated by an enhanced resistance of tuber tissue cell walls to Erwinia -derived enzymes, an increased PPO- and PAL-activity in tuber tissue as well as by a strengthened formation of necrosis on the wound surface of tubers after infection with Ec -bacteria.     

Analysis of protease activity in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis> on peanut seed infection and aflatoxin contamination

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.     

The G-protein subunits FGA2 and FGB1 play distinct roles in development and pathogenicity in the banana fungal pathogen Fusarium oxysporum f. sp. cubense

The soil-borne fungus Fusarium oxysporum f. sp. cubense causes banana (Musa spp.) vascular wilt. Here, we examine the roles of G-protein α and β subunit genes fga2 and fgb1 in F. oxysporum development and pathogenicity. Deletion of either or both genes led to increased heat resistance, lower cAMP levels, and enhanced pigmentation, whereas phenotypic defects of colony morphology and reduced conidiation were seen in Δfgb1 and Δfga2/Δfgb1 deletion strains but not in Δfga2. Conversely, Δfgb1 retained greater virulence against banana, suggesting that FGA2 regulates fungal virulence whereas FGB1 modulates both development and virulence, potentially via the cAMP-dependent protein kinase A pathway.     

Efficacy of certain agrochemicals on Aspergillus spp. and subsequent aflatoxin production in rice

Efficacy of certain fungicides and non-conventional chemicals against Aspergillus spp. contamination and subsequent aflatoxin production in rice was investigated. Among the 10 fungicides tested, carbendazim, contaf plus, folicur, propiconazole and saaf completely inhibited the growth of all Aspergillus spp. and aflatoxin B₁ (AFB1) production at 1 g or ml/kg concentration. Of the five non-conventional chemicals tested, benzoic acid effectively inhibited the mycelial growth of Aspergillus flavus (72%) at 4 g/kg, completely inhibited the Aspergillus parasiticus and Aspergillus niger even at 1 g/kg and Aspergillus ochraceus at 4 g/kg concentration. Vanillin completely reduced the AFB1 production at 4 g/kg of seed followed by sodium chloride with out inhibiting the mycelial growth. This study reveals that fungicides and non-conventional chemicals had effectively inhibited the mycelial growth of Aspergillus spp. and AFB1 production in rice.     

Diaporthe sclerotioides exhibits no host specificity among cucurbit species

Plant–fungal specificity between cucurbitaceous crops and Diaporthe sclerotioides, the causal agent of black root rot, was studied using cucumbers (Cucumis sativa), melons (Cucumis melo), pumpkins (Cucurbita maxima), watermelons (Citrullus lanatus) and bottlegourd (Lagenaria siceraria var. gourda). Twelve D. sclerotioides isolates from these cucurbit species were cross‐inoculated. The virulence of the isolates was evaluated as the area under the disease progress curve (AUDPC). All cucurbit species were susceptible to each isolate, but AUDPCs were significantly different among the hosts, with the order of greatest to least being melon, cucumber, watermelon, bottlegourd and finally, pumpkin. The infectiveness of isolates was assessed as the quantity of D. sclerotioides DNA detected in the hypocotyls of seedlings 2 weeks after inoculation using a real‐time PCR protocol. The fungal DNA quantities varied among the species in the same order as the AUDPCs. Whilst there were statistically significant correlations between the virulence and infectiveness of D. sclerotioides isolates in cucumbers, melons and bottlegourds, their coefficients of determination were not high (r² < 0·6). Orthogonal contrasts indicated no specificity in either the fungal virulence or infectiveness between D. sclerotioides isolates and the cucurbit hosts from which these isolates originated. Thus, although the degree of host susceptibility to D. sclerotioides varies among cucurbit species, the absence of specificity to the host species in either virulence or infectiveness suggests the pathogen may spread via various cucurbit crops, irrespective of their original host species.     

A survey of mold flora and of the mycotoxin deoxynivalenol in corn imported to ISRAEL

Shipments of corn imported to Israel from the U.S.A. during 1985 were sampled during unloading and examined for the presence of fungi and the trichothecene deoxynivalenol (DON). The most frequent species found on the corn wasAspergillus flavus followed byA. versicolor; three otherAspergilli — A. niger, A. ochraceus andA. fumigatus — were found at a much lower frequency. DON analysis was performed by electron capture detection gas chromatography after derivatization with heptafluorobutyryl imidazole. At fortification levels of 0.1, 1 and 2 μg/g, recoveries of 80—100% were obtained. Although DON was not detected in any of the samples analyzed, the observation (in the shipments) of fungi belonging to the generaFusarium andAspergillus suggests a potential danger of mycotoxin contamination. The relatively high moisture content of the samples increased the likelihood of such a hazard.     

Impact of <Emphasis Type="Italic">Dysdercus koenigii</Emphasis> Fabricius (Hemiptera: Pyrrhcoridae) density-dependent population on agronomic and qualitative characteristics of different transgenic cotton varieties

The cotton stainer, Dysdercus koenigii Fabricius (Hemiptera: Pyrrhcoridae), has become a major threat to transgenic cotton as it causes warts on the internal carpel wall of cotton boll, severe lint staining, lint locks, and lint lesions. Thus, keeping in view the importance of this pest on cotton, in the present study, screening of 13 transgenic cotton genotypes was performed and the population of D. koenigii was determined on these genotypes during 2012–13. Furthermore, among these genotypes, a high yielding advanced cultivar (FH-114) was selected for further experiments. A number of five different densities of D. koenigii (5, 10, 15, 20, and 25) at adult stage with well-developed proboscis were released in the cages along with a control treatment. Cotton genotypes FH-312, FH-2073, FH-Lalazar, FH-142, and MNH-886 possessed minimum population (1.33 D. koenigii per plant) compared to FH-324 and FH-444 (6.0 D. koenigii per plant). The number of bolls per plant and boll weight decreased as a result of increased densities. The increased density of D. koenigii resulted in fewer bolls per plant (12.1 bolls), reduced boll weight (2.1 g), germination (39.1%), ginning out turn (38.3%), staple length (27.8 mm), and staple fineness (4.0 μg/in.) when compared with un-infested plants (30.6, 3.2 g, 77.3%, 41.5%, 28.8 mm, and 4.4 μg/in.), respectively. Correlation analysis revealed that bolls per plant, boll weight, germination, ginning out turn, and staple fineness resulted in negative and significant correlation with density of D. koenigii with r-values of ?0.95, ?0.98, ?0.98, ?0.8, and ?0.85, respectively. Coefficient of determination (R²) demonstrated that bolls per plant, boll weight, germination, ginning out turn, staple length, and staple fineness contributed with 91, 96, 96, 79, 43 and 73%, respectively, to the total variability at different densities of D. koenigii. Our findings demonstrated that D. koenigii has become potential major pest and causes quantitative and qualitative losses to transgenic cotton, hence, there is a need to develop appropriate control measures for controlling its population to avoid further losses.