Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins. The GFP strain and the wild-type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight. GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus. The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus. For example, the inner seed coat and cotyledon were colonized by the fungus within 72 h of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds. The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production. The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding. In addition, development of GFP expressing A. flavus strain provides an easy and rapid assay procedure for studying the ecology, etiology, and epidemiology of cotton boll rot caused by A. flavus resulting in aflatoxin contamination. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

The Degree of Susceptibility of Nectary-Inoculated Cotton Flowers and Bolls to Subsequent Seed Infection by Aspergillus flavus Is Determined at or before Anthesis

In greenhouse and field studies, cotton (Gossypium hirsutum L.) flowers were inoculated with Aspergillus flavus at the involucral nectaries. Bolls developing from early-season flowers had significantly higher percentages of A. flavus-infected seed than did bolls from flowers formed later in the season. Seeds from bolls inoculated 2 weeks after anthesis had the same infection levels as those from flowers inoculated at anthesis. These results indicate that early-season flowers are predisposed to A. flavus infection and that the degree of susceptibility at anthesis is retained through early boll development.     

Transmission of Alternaria macrospora in Cotton Seeds

Alternaria macrospora was isolated from seeds only after the natural opening of the bolls and exposure of seeds to an environment in which the fungus was present. The fungus lacks the ability to penetrate the boll wall and reach the seed site. Attempts to isolate the pathogen from seeds of immature bolls at different developmental stages failed. Internal infection by slow injection resulted in seed infection and partial shedding of the injected plant parts which was high in buds and decreased with the ripening to mature bolls. Severity of seed infection was not dependent on either inoculum level, boll physiological age or even if the boll itself was not diseased. Infection of flowers under field conditions caused flower shedding. Naturally infected seeds or inoculated seeds with inoculum levels of 100 spores/ml and above resulted in diseased cotyledons, the incidence of which was, for inoculated seeds, positively correlated with inoculum level. A small difference was observed between cultivars in susceptibility to artificial inoculation at the cotyledon stage. A. macrospora survived on commercial cotton seeds and on post-season plants left growing at the field edges. Survival in plant debris under field conditions was minimal and may only have a minor effect on field reinfestation.     

Research on the aflatoxin problem in groundnut at ICRISAT

Summary Aflatoxin contamination of groundnut is a serious problem in most groundnut producing countries and as such is given high research priority by the Groundnut Improvement Program of ICRISAT. Since 1979 we have concentrated on selecting cultivars resistant to seed invasion and colonization by toxigenicAspergillus flavus, and/or to aflatoxin production following invasion by the fungus. Resistance to invasion and colonization byA. flavus of rehydrated, mature seed has been found, and confirmed, in some cultivars. We have also screened several groundnut cultivars for seed resistance in the field, both under natural conditions and with the inoculum of the fungus added to the soil in the pod zone. Some cultivars with resistance to seed colonization also showed resistance to seed invasion byA. flavus. None of the cultivars tested has shown complete resistance to aflatoxin production but significant cultivar differences occurred in the amounts of aflatoxin produced in seeds inoculated with a toxigenic strain ofA. flavus.ICRISAT Journal Article No. JA-316     

Preharvest aflatoxin contamination of maize inoculated withAspergillus flavus andFusarium moniliforme

A two-year factorial experiment was utilized to test plants field-inoculated singly and in combination withAspergillus flavus andFusarium moniliforme. Pinbar inoculations were made through the husks with conidial suspensions, and 10-ear maize samples were harvested at 60 days post-silking for aflatoxin determinations. When ears were inoculated with both fungi simultaneously,F. moniliforme reduced aflatoxin formation byA. flavus isolate NRRL 3357 by approximately two-thirds.F. moniliforme had no significant effect on naturally occurring aflatoxin contamination byA. flavus. This may be due to the timing of infection by both fungi in the field. In nature,A. flavus andF. moniliforme respond differently to the environment, offering one explanation of whyF. moniliforme did not measurably affect the other fungus.     

Separate and combined applications of nontoxigenic Aspergillus flavus and A. parasiticus for biocontrol of aflatoxin in peanuts

A 2-year study was carried out to determine the effect of applying nontoxigenic strains of Aspergillus flavus and A. parasiticus to soil separately and in combination on preharvest aflatoxin contamination of peanuts. A naturally occurring, nontoxigenic strain of A. flavus and a UV-induced mutant of A. parasiticus were applied to peanut soils during the middle of each of two growing seasons using a formulation of conidia-coated hulled barley. In addition to an untreated control, treatments included soil inoculated with nontoxigenic A. flavus only, soil inoculated with nontoxigenic A. parasiticus only, and soil inoculated with a mixture of the two nontoxigenic strains. Plants were exposed to late-season drought conditions that were optimal for aflatoxin contamination. Results from year one showed that significant displacement (70%) of toxigenic A. flavus occurred only in peanuts from plots treated with nontoxigenic A. flavus alone; however, displacement did not result in a statistically significant reduction in the mean aflatoxin concentration in peanuts. In year two, soils were re-inoculated as in year one and all treatments resulted in significant reductions in aflatoxin, averaging 91.6%. Regression analyses showed strong correlations between the presence of nontoxigenic strains in peanuts and aflatoxin reduction. It is concluded that treatment with the nontoxigenic A. flavus strain alone is more effective than the A. parasiticus strain alone and equally as effective as the mixture. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Effects of methyl jasmonate on phytoalexin production and aflatoxin control in the developing cotton boll

Artificially wounded 22–27-day old developing cotton bolls were initially inoculated with, (1) a cell-free, hot water-soluble mycelial extract (CFME) of an atoxigenic strain of Aspergillus flavus or with, (2) chitosan lactate (CHL) or with, (3) CFME or CHL and then exposed to gaseous methyl jasmonate (MJ) or, (4) exposed to MJ alone. Five days after these treatments, the induction of the sesquiterpenoid naphthol phytoalexins, 2,7-dihydroxycadalene (DHC) and 2-hydroxy-7-methoxy cadalene (HMC), lacinilene C, lacinilene C7-methyl ether, and the coumarin phytoalexin-scopoletin was determined on the excised carpel discs surrounding the inoculated surfaces of the developing cotton bolls. The results indicated a two- or three-fold increase in the production of the phytoalexins when gaseous MJ was added in combination to the CFME or the CHL elicitors. In a separate experiment, 22–27-day old developing cotton bolls were pretreated for a five-day period as described above and then a spore suspension of a toxigenic strain of A. flavus was introduced into a second artificial wound which was produced adjacent to the first wound. On boll maturity, the cottonseeds located within the locules underlying the areas that were pretreated with both elicitors and MJ then later infected with toxigenic A. flavus exhibited a 75–95% aflatoxin B₁ inhibition. These results suggest a host defense mechanism which may be triggered by both elicitors and MJ.     

Cytochemical localization and ultrastructure of Aspergillus flavus in cottonseed

Cottonseeds having fluorescent fibers were harvested from fields in Arizona and examined utilizing light microscopy and transmission electron microscopy. The occurrence of fluorescent fibers indicated that seeds had been infected by Aspergillus flavus during development. Presence of A. flavus was verified by plating portions of seeds with fluorescent fibers. Hyphae, conidial heads, and conidia were identified readily in differentially-stained cotyledon tissue processed for light microscopy. Utilization of transmission electron microscopy permitted observations on lignified seed coats and cotyledons of mature cottonseeds. Hyphae were located throughout the cotyledon and in the nonlignified layers of the seed coat. The identification of hyphae in cross sections of vessel elements within the seed coat provided ultrastructural evidence supporting the hypothesis that A. flavus may enter seeds via the vascular tissue. Controls for the microscopy studies included observations on cottonseeds with no visual signs of infection and on laboratory-grown cultures of A. flavus. These observations demonstrated that the hyphae localized within fluorescent seeds had features characteristic of A. flavus and that fungal-like structures do not occur within uninfected seeds.     

Nectaries as Entry Sites for Aspergillus flavus in Developing Cotton Bolls

Cotton flowers in replicate plots in two fields near Phoenix, Ariz., were tagged in June at the beginning of the flowering period. Flowers or developing bolls from these tagged flowers were inoculated on the involucral (bracteal) nectaries with dry spores of Aspergillus flavus. The bolls were harvested as they matured in August, and the seeds were assessed for the presence of the fungus. The number of infected seed from flowers and bolls inoculated up to 25 days after anthesis was significantly higher than that in uninoculated controls. Seeds from bolls inoculated after 25 days postanthesis did not differ significantly from controls in degree of infection. We postulate that the sharp decline in the ability of the fungus to infect the plant and seed is a result of physical or biochemical changes in the boll as it reaches physiological maturity or biochemical changes in the entire plant as it develops.     

Preharvest seed infection byAspergillus flavus group fungi and subsequent aflatoxin contamination in groundnuts in relation to soil types

Preharvest seed infection byAspergillus flavus and aflatoxin contamination in selected groundnut genotypes (fourA. flavus-resistant and fourA. flavus-susceptible) were examined in different soil types at several locations in India in 1985–1990. Undamaged mature pods were sampled at harvest and seed examined forA. flavus infection and aflatoxin content in two or more trials at ICRISAT Center on light sandy soils and red sandy loam soils (Alfisols), and on Vertisols, at Anantapur on light sandy soils, and at Dharwad and Parbhani on Vertisols. Rainy season trials (1985–1989) were all rainfed. Post-rainy season trials were irrigated; late-season drought stress (90 days after sowing (DAS) until harvest at 125 DAS) was imposed in the 1987/88 and 1989/90 seasons.A. flavus infection and aflatoxin contamination levels were much lower in seed of all genotypes from Vertisols than in seed from Alfisols across locations and seasons. Vertisols also had significantly lower populations ofA. flavus than Alfisols. There were no marked differences between light sandy soils and red sandy loam soils (Alfisols) in respect of seed infection byA. flavus and aflatoxin contamination. Significant interactions between genotypes and soil types were evident, especially in theA. flavus-susceptible genotypes. Irrespective of soil types,A. flavus-resistant genotypes showed lower levels of seed infection byA. flavus and other fungi than didA. flavus-susceptible genotypes. The significance of the low preharvest aflatoxin risk in groundnuts grown on Vertisols is highlighted.ICRISAT Journal Article No. JA 1122     

Pathogenic aspects of Pantoea agglomerans in relation to cotton boll age and Dysdercus cingulatus (Fabricius) transmitting seed and boll rot in cotton germplasm

Bacterial seed and boll rot is a newly emerging cotton disease in Pakistan. Twenty-one cotton varieties were screened to find resistance source against the disease. None of these was found to be resistant. Five cotton varieties (CIM-595, MK2, BT-986, BT-986 & SG-1) having 700–1400 Area under disease progress curve (AUDPC) units were found to be moderately resistant to the disease. SLH-317, FH-942, BT-222, BT-666, MNH-457 ranging from 1401–1700 AUDPC units were moderately susceptible while MNH-456, SLH-336, 9811, FH-942, MNH-886 susceptible to boll rot. Seven varieties (FH-114, FH-113, BT-7, BT-212, SLH-BT-4, BT-212 and FH-941) were highly susceptible to bacterial seed and boll rot indicated by 2001–2300 AUDPC units. Biochemical tests identified bacterial isolates as Pantoea agglomerans. Different inoculation techniques were assessed for bacterial pathogenicity and symptoms of boll rot were only observed in needle punctured bolls. One, two and three weeks old bolls were mechanically inoculated by injecting bacterial suspension to evaluate the boll’s age impact on disease severity. Maximum severity was observed in two weeks old bolls. Red cotton bugs (Dysdercus cingulatus) were fed on artificially inoculated diseased bolls and then transferred on healthy bolls. Diseased symptoms were noticed on healthy cotton bolls. Bacterial colonies were recovered and red cotton bug was confirmed as the disease-transmitting vector.     

Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

The fungal entomopathogen Beauveria bassiana became established as an endophyte in coffee seedlings grown in vitro and inoculated with B. bassiana suspensions in the radicle. The fungus was recovered as an endophyte 30 and 60 days postinoculation, from stems, leaves, and roots, and at 60 days postinoculation one of the isolates was also recovered as an epiphyte. Fusarium sp., Rhodotorula sp., and four bacterial morpho-species were also detected, indicating these were present as endophytes in the seed.     

Evaluation of selected geocarposphere bacteria for biological control of Aspergillus flavus in peanut

Selected bacterial strains isolated from the region of peanut pod development (geocarposphere) and two additional bacterial strains were screened as potential biological control agents against Aspergillus flavus invasion and subsequent aflatoxin contamination of peanut in laboratory, greenhouse, and field trials. All 17 geocarposphere strains tested delayed invasion of young roots and reduced colonization by the fungus in a root-radicle assay used as a rapid laboratory prescreen. In a greenhouse study, seven bacterial strains significantly reduced pod colonization by A. flavus compared to the control. In a field trial, conducted similarly to the greenhouse assay, pods sampled at mid-peg from plants seed-treated with suspensions of either 91A-539 or 91A-550 were not colonized by A. flavus, and the incidence of pods invaded from plants treated with either 91A-539 or 91A-599 was consistently lower than nonbacterized plants at each of five sampling dates. At harvest, 8 geocarposphere bacterial strains significantly lowered the percentage of pods colonized (> 51%) compared to the control. Levels of seed colonization ranged from 1.3% to 45% and did not appear related to aflatoxin concentrations in the kernels.     

Line x tester analysis for fixed effect model in cotton (Gossypium hirsutum L.)

Summary The data from an experiment in cotton consisting of three testers and 12 lines selected deliberately have been analysed. The investigation showed higher specific combining ability variance for yield of seed cotton and number of bolls, indicating the predominance of non-additive gene action. Of parental lines, H777 was found to possess high g.c.a. effects for seed cotton yield, number of bolls and number of sympodes. Parent H842 contributed only for boll weight, whereas H655 was good general combiner for number of monopodes. There appeared to be better chances for increasing the yield by exploiting hybrid vigour for the number of bolls and boll weight. The presence of marked non-additive gene effects, in addition to additive gene effects, indicated the need for exploiting both the fixable and non-fixable components of genetic variance for increasing productivity in cotton.     

拟南芥ERD15基因缺失突变体的分子鉴定和表型分析

为探究ERD15基因功能,利用反向遗传学,通过PCR及半定量PCR筛选鉴定出拟南芥(Arabidopsis thaliana) ERD15基因的T-DNA插入纯合突变体,并对其表型进行观察分析。结果表明,erd15突变体莲座叶数目显著增多,提前3～4 d开花,突变体比野生型更早从营养生长转向生殖生长。拟南芥野生型植株主茎为圆柱体,平均直径1.29 mm,而erd15突变体主茎扁平,平均直径达到2.27mm,具极显著差异。与野生型相比,erd15突变体果实心皮发育受到影响,隔膜上排列有多排种子,果荚顶端膨大,长度缩短37.67%,但角果平均结籽数升高。因此,ERD15基因参与了调控拟南芥植株的生殖生长过程。     

Effect ofAspergillus parasiticus soil inoculum on invasion of peanut seeds

Environmental control plots adjusted to late season drought and elevated soil temperatures where inoculated at peanut planting with low and high levels of conidia, sclerotia, and mycelium from a brown conidial mutant ofAspergillus parasiticus. Percentage infection of peanut seeds from undamaged pods was greatest for the subplot containing the high sclerotial inoculum (15/cm² soil surface). Sclerotia did not germinate sporogenically and may have invaded seeds through mycelium. In contrast, the mycelial inoculum (colonized peanut seed particles) released large numbers of conidia into soil. Soil conidial populations of brownA. parasiticus from treatments with conidia and mycelium were positively correlated with the incidence of seed infection in undamaged pods. The ratio ofA. flavus to wild-typeA. parasiticus in soil shifted from 7:3 to 1:1 in the uninoculated subplot after instigation of drought, whereas in all subplots treated with brownA. parasiticus, the ratio of the two species became approximately 8:2. Despite high levels of brownA. parasiticus populations in soil, nativeA. flavus often dominated peanut seeds, suggesting that it is a more aggressive species. Sclerotia of wild-typeA. parasiticus formed infrequently on preharvest peanut seeds from insect-damaged pods.     

带叶兜兰种子原地共生萌发及有效菌根真菌的分离与鉴定

为获得带叶兜兰(Paphiopedilum hirsutissimum)种子萌发的共生真菌,采用原地共生萌发技术获得了2株自然萌发的小幼苗,并分离和筛选出了有效的种子萌发共生菌——瘤菌根菌(Epulorhiza sp.)。为验证分离菌株对带叶兜兰种子萌发的有效性,将Phs34号菌株与带叶兜兰种子在灭菌后的原生境基质上进行室内共生萌发试验,结果表明,经过6周的培养,对照组没有观察到种子的萌发;接菌的种子胚明显膨大,突破种皮,形成原球茎,平均萌发率为(58.35±3.41)%。这表明分离得到的瘤菌根菌能促进带叶兜兰的种子萌发。     

<Emphasis Type="Italic">Aspergillus flavus</Emphasis> hydrolases: their roles in pathogenesis and substrate utilization

Aspergillus flavus is a fungus that principally obtains resources for growth in a saprophytic mode. Yet, it also possesses the characteristics of an opportunistic pathogen with a wide, non-specific host range (plants, animals, and insects). It has attained a high level of agricultural significance due to production of the carcinogen aflatoxin, which significantly reduces the value of contaminated crops. To access a large variety of nutrient substrates and penetrate host tissues, A. flavus possesses the capacity to produce numerous extracellular hydrolases. Most work on A. flavus hydrolases has focused on the serine and metalloproteinases, pectinase P2c, and amylase. Many hydrolases are presumed to function in polymer degradation and nutrient capture, but the regulation of hydrolase secretion is complex and substrate dependent. Proteinases are employed not only to help access protein substrates, such as elastin that is found in mammals and insects, but may also play roles in fungal defense and virulence. Secretion of the endopolygalacturonase P2c is strongly correlated with isolate virulence (against plants) and maceration of cotton boll tissues. In some hosts, secretion of α-amylase is critical for starch digestion and may play a critical role in induction of aflatoxin biosynthesis. Despite a significant body of work, much remains to be learned about hydrolase production and utilization by A. flavus. This information may be critical for the formulation of successful strategies to control aflatoxin contamination in affected commodities.     

Effect of kind and method of fungicidal treatment of bean seed on infections by the VA-mycorrhizal fungus Glomus macrocarpum and by the pathogenic fungus Fusarium solani

To test the effect of seed treatment with fungicides on the development of mycorrhizal fungi, bean seeds were treated with fungicide dry or vehicled in the organic solvents, ethanol or dichloromethane and then planted in soil inoculated with the mycorrhizal fungus Glomus macrocarpum and/or the plant pathogenic fungus Fusarium solani. Measurements were made at 4 day intervals, to evaluate the location and extent of colonization of either Glomus macrocarpum or Fusarium solani in the root system. Most combinations of fungicide-solvent had little effect on the extent of colonization by each fungus individually. However, when both fungi were inoculated together, symptoms of F. solani were seen only in the tips of roots which indicate that the mycorrhizal fungus was able to limit the occurrence of the pathogenic fungus.     

Biological Interactions to Select Biocontrol Agents Against Toxigenic Strains of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> from Maize

Biological control represent an alternative to the use of pesticides in crop protection. A key to progress in biological control to protect maize against Fusarium verticillioides and Aspergillus flavus maize pathogens are, to select in vitro, the best agent to be applied in the field. The aim of this study was to examine the antagonistic activity of bacterial and yeast isolates against F.verticillioides and A. flavus toxigenic strains. The first study showed the impact of Bacillus amyloliquefaciens BA-S13, Microbacterium oleovorans DMS 16091, Enterobacter hormomaechei EM-562T, and Kluyveromyces spp. L14 and L16 isolates on mycelial growth of two strains of A. flavus MPVPA 2092, 2094 and three strains of F. verticillioides MPVPA 285, 289, and 294 on 3% maize meal extract agar at different water activities (0.99, 0.97, 0.95, and 0.93). From this first assay antagonistics isolates M. oleovorans, B. amyloliquefaciens and Kluyveromyces sp. (L16) produced an increase of lag phase of growth and decreased a growth rate of all fungal strains. These isolates were selected for futher studies. In vitro non-rhizospheric maize soil (centrally and sprayed inoculated) and in vitro maize (ears apex and base inoculated) were treated with antagonistics and pathogenic strains alone in co-inoculated cultures. Bacillus amyloliquefaciens significantly reduced F. verticillioides and A. flavus count in maize soil inoculated centrally. Kluyveromyces sp. L16 reduced F. verticillioides and A. flavus count in maize soil inoculated by spray. Kluyveromyces sp. L16 was the most effective treatment limiting percent infections by F. verticillioides on the maize ears.