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.     

A possible relationship between phytoalexin production in the cotton leaf and a phytotoxic response

A 10-day time-course study on the production of 2,7-dihydroxycadalene, 2-hydroxy-7-methoxycadalene, lacinilene C and lacinilene C 7-methyl ether in cotton leaves induced by cell-free mycelial extracts of Aspergillus flavus showed that the cadalenes and the lacinilenes accumulate in a cyclic fashion. The initial increase at 2 days is followed by a greater increase at 6 days after treatment. The location of these compounds was found predominately either in a 6 mm wounded, treated area or in a 3 mm area immediately surrounding the 6 mm treated area of the leaf. Lacinilene C and lacinilene C 7-methyl ether were both phytotoxic in a Lemna minor bioassay. Endogenous constituents produced by plant cell damage could have triggered the production of the cadalenes and lacinilenes observed.     

The occurrence of Aspergillus flavus in vegetative tissue of cotton plants and its relation to seed infection

Twenty-seven mature cotton bolls with Aspergillus flavus Link colonies naturally occurring on the surface of the boll or lint were collected in the field in Arizona along with their subtending stems and peduncles. Bolls inoculated through the carpel wall 30 days after anthesis were allowed to mature in the field and were collected in the same manner. The seed and stem and peduncle sections of each boll were surface-sterilized, plated on agar media and observed for A. flavus. Seventy-eight percent of the naturally contaminated bolls with A. flavus in the seed also had the fungus in the stem and peduncle, whereas only 31% of the naturally contaminated bolls with no A. flavus in the seed had the fungus in the stem or peduncle. This difference was significant (P=0.0125), indicating a positive relationship between seed infection and stem and peduncle infection. All of the bolls inoculated through the carpel wall had A. flavus in the seed, but only 11% of the stem and peduncle sections were infected, indicating that the fungus does not readily grow downward from the boll into the supporting stem or peduncle.This unidirectional pattern of movement (upward) was further substantiated in greenhouse experiments where cotton seedlings were inoculated at the cotyledonary leaf scar with A. flavus and plants were sequentially harvested, surface sterilized and plated. Aspergillus flavus was isolated from the cotyledonary leaf scar, flower buds, developing bolls, and stem sections in the upper portion of the plant. It was never isolated from roots or stem sections below the cotyledonary node, again indicating that the fungus does not readily move downward through the plant.     

Light filtering by epidermal flavonoids during the resistant response of cotton to Xanthomonas protects leaf tissue from light-dependent phytoalexin toxicity

2,7-Dihydroxycadalene and lacinilene C, sesquiterpenoid phytoalexins that accumulate at infection sites during the hypersensitive resistant response of cotton foliage to Xanthomonas campestris pv. malvacearum, have light-dependent toxicity toward host cells, as well as toward the bacterial pathogen. Adaxial epidermal cells surrounding and sometimes covering infection sites turn red. The red cells exhibited 3-4-fold higher absorption at the photoactivating wavelengths of sunlight than nearby colorless epidermal cells. Red epidermal cells protected underlying palisade mesophyll cells from the toxic effects of 2,7-dihydroxycadalene plus sunlight, indicating a role for epidermal pigments in protecting living cells that surround infection sites from toxic effects of the plant’s own phytoalexins. A semi-quantitative survey of UV-absorbing substances extracted from epidermal strips from inoculated and mock-inoculated cotyledons indicated that the principal increase in capacity to absorb the photoactivating wavelengths was due to a red anthocyanin and a yellow flavonol, which were identified as cyanidin-3-O-β-glucoside and quercetin-3-O-β-glucoside, respectively.     

Variability among atoxigenic Aspergillus flavus strains in ability to prevent aflatoxin contamination and production of aflatoxin biosynthetic pathway enzymes.

Five strains of Aspergillus flavus lacking the ability to produce aflatoxins were examined in greenhouse tests for the ability to prevent a toxigenic strain from contaminating developing cottonseed with aflatoxins. All atoxigenic strains reduced contamination when inoculated into developing bolls 24 h prior to the toxigenic strain. However, only one strain, AF36, was highly effective when inoculated simultaneously with the toxigenic strain. All five strains were able to inhibit aflatoxin production by the toxigenic strain in liquid fermentation. Thus, in vitro activity did not predict the ability of an atoxigenic strain to prevent contamination of developing bolls. Therefore, strain selection for competitive exclusion to prevent aflatoxin contamination should include evaluation of efficacy in developing crops prior to field release. Atoxigenic strains were also characterized by the ability to convert several aflatoxin precursors into aflatoxin B1. Four atoxigenic strains failed to convert any of the aflatoxin biosynthetic precursors to aflatoxins. However, the strain (AF36) most effective in preventing aflatoxin contamination in developing bolls converted all tested precursors into aflatoxin B1, indicating that this strain made enzymes in the aflatoxin biosynthetic pathway.     

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.     

Aflatoxigenic Isolates of Aspergillus flavus from Pecans

Of 120 isolates of the Aspergillus flavus group from pecans used in bakery products, 85 were shown to produce aflatoxin on yeast extract sucrose medium. Extracts from moldy sections of raw pecans obtained commercially at the retail level showed aflatoxin-like spots on thin-layer chromatography. Cooked (autoclaved) pecans inoculated with toxigenic isolates of A. flavus were also good substrates for aflatoxin production.     

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.     

RNAi suppression of CYP82D P450 hydroxylase,an enzyme involved in gossypol biosynthesis,enhances resistance to Fusarium wilt in cotton

Cotton plants produce two classes of terpenoid defence compounds against pathogens and other pests. Both classes are derived from a common sesquiterpenoid precursor, δ-cadinen-2-one, which enters either the gossypol pathway or the lacinilene pathway. Blocking the gossypol pathway by RNAi suppression of the early pathway biosynthetic enzyme CYP82D hydroxylase resulted in enhanced resistance to the Fusarium wilt pathogen. Analyses of root terpenoids revealed no overall increases in the products of the gossypol pathway in the roots infected by the wilt pathogen. However, the lacinilene pathway was elicited by the pathogen and the lacinilene levels were 19-fold higher in the RNAi plants than in wild-type plants. In the pathogen inoculated RNAi 73R plants, the concentrations of DHC and HMC were 231 μg and 886 μg/g dry roots, respectively, which may have contributed to the inhibition of fungal invasion. In comparison, the concentrations of DHC and HMC in the pathogen inoculated control wild-type 73W plants were only 0.7 μg and 58 μg/g dry roots, respectively. Fungitoxicity testing showed that DHC at 100 μg/ml inhibited growth of the Fusarium wilt pathogen by >93%. Treatment with the phytohormone jasmonic acid failed to elicit production of lacinilene pathway terpenoids in roots of either RNAi plants or their wild-type sibling lines, but increased production of gossypol pathway terpenoids with concentrations in RNAi plants 80%–97% less than those in wild-type plants. This indicates that induction of the lacinilene pathway is not directly mediated by jasmonic acid signalling and requires other signalling to activate the pathway. These results illustrate possible mechanisms of wilt disease resistance in cotton and provide a new approach to increase host resistance by manipulating these two major cotton chemical defence pathways.     

Investigation of the mechanism of phytoalexin accumulation in soybean induced by glucan or mercuric chloride

Soybean cotyledons which had been treated with glucan from Phytophthora megasperma f.sp. glycinea or with mercuric chloride were pulse-labeled with ¹⁴CO₂ and then the ¹⁴C-incorporation into the phytoalexins was determined. The kinetics of ¹⁴C-incorporation into phytoalexins (glyceollin isomers and 3,6α,9-trihydroxypterocarpan) was very similar with the two types of elicitors. Metabolic rates of phytoalexins were determined by pulse-chase experiments. The apparent half-life of metabolism was about 100 h for glyceollin with either glucan or HgCl₂. The half-lives for trihydroxypterocarpan were 39 h with glucan and 14 h with HgCl₂. According to our results levels of glyceollins in soybean cotyledons are mainly controlled by their rates of synthesis. Biotic (glucan) and abiotic (HgCl₂) elicitors have similar induction effects. Both types of elicitors could act by effecting the release of endogenous elicitors.     

Presence of Aspergillus flavus in developing cotton bolls and its relation to contamination of mature seeds.

Aspergillus flavus spores were dusted onto the involucral nectaries of cotton flowers. The fungus was present in 20 to 58% of the immature bolls harvested 25 or 35 days after anthesis. Among similarly inoculated bolls fully matured either in the field or under sterile conditions at ambient temperatures after excision from the plants, only 3 to 14% contained A. flavus in the seeds. There was no significant difference in the numbers of contaminated bolls between the excised and field-matured treatments. It is concluded that A. flavus is present in developing cotton bolls before dehiscence, but its presence does not ensure infection of mature seeds, and that excision does not reduce A. flavus contamination if the bolls are maintained at ambient temperatures.     

Presence of Aspergillus flavus in developing cotton bolls and its relation to contamination of mature seeds

Aspergillus flavus spores were dusted onto the involucral nectaries of cotton flowers. The fungus was present in 20 to 58% of the immature bolls harvested 25 or 35 days after anthesis. Among similarly inoculated bolls fully matured either in the field or under sterile conditions at ambient temperatures after excision from the plants, only 3 to 14% contained A. flavus in the seeds. There was no significant difference in the numbers of contaminated bolls between the excised and field-matured treatments. It is concluded that A. flavus is present in developing cotton bolls before dehiscence, but its presence does not ensure infection of mature seeds, and that excision does not reduce A. flavus contamination if the bolls are maintained at ambient temperatures.     

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.     

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.     

Lack of Host Specialization in Aspergillus flavus

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22°C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton bolls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.     

Changes in cotton leaf chemistry induced by volatile elicitors

Cotton (Gossypium hirsutum) leaves were exposed for 7 days to volatile chemicals originating from Aspergillus flavus-infected cotton leaves, A. flavus cultures or mechanically damaged cotton leaves. Volatiles from A. flavus-infected leaves triggered significant increases of 52 and 34% in phloroglucinol-reactive compounds in wounded or undamaged cotton leaves, respectively. Increased production of heliocides (C₂₅ terpenoid aldehydes) were found in the volatile recepient wounded or undamaged cotton leaves. The heliocides are natural insecticides presumed localized in the subepidermal pigment glands in leaves. Myrcene, a volatile precursor of heliocide H₂, also caused significant increases in heliocide production when leaves were exposed to the volatilized chemical.     

Lipid peroxidation is a consequence of elicitor activity

Elicitor-active preparations from the fungal pathogen of bean Colletotrichum lindemuthianum stimulated the accumulation of products characteristic of lipid peroxidation in treated bean tissues. Bean suspension cells treated with crude and purified elicitors accumulated `lipofuscin-like pigment' (LEP) and malondialdehyde. The accumulation of LFP after about 6 h of treatment coincided with the onset of visible browning and production of the bean phytoalexins kievitone, phaseollin, and phaseollinisoflavan. The induction of phytoalexins and accumulation of LFP were also triggered by treatments with generators of activated oxygen species, xanthine:xanthine oxidase and Fe:ethylenediaminedi-o-hydroxyphenylacetic acid. These data suggest that generation of active oxygen species may be involved in lipid peroxidation triggered by elicitors.     

VdNEP, an Elicitor from Verticillium dahliae, Induces Cotton Plant Wilting

Verticillium wilt is a vascular disease of cotton. The causal fungus, Verticillium dahliae, secretes elicitors in culture. We have generated ~1,000 5′-terminal expressed sequence tags (ESTs) from a cultured mycelium of V. dahliae. A number of ESTs were found to encode proteins harboring putative signal peptides for secretion, and their cDNAs were isolated. Heterologous expression led to the identification of a protein with elicitor activities. This protein, named V. dahliae necrosis- and ethylene-inducing protein (VdNEP), is composed of 233 amino acids and has high sequence identities with fungal necrosis- and ethylene-inducing proteins. Infiltration of the bacterially expressed His-VdNEP into Nicotiana benthamiana leaves resulted in necrotic lesion formation. In Arabidopsis thaliana, the fusion protein also triggered production of reactive oxygen species and induced the expression of PR genes. When added into suspension cultured cells of cotton (Gossypium arboreum), the fusion protein elicited the biosynthesis of gossypol and related sesquiterpene phytoalexins at low concentrations, and it induced cell death at higher concentrations. On cotton cotyledons and leaves, His-VdNEP induced dehydration and wilting, similar to symptoms caused by a crude preparation of V. dahliae elicitors. Northern blotting showed a low level of VdNEP expression in the mycelium during culture. These data suggest that VdNEP is a wilt-inducing factor and that it participates in cotton-V. dahliae interactions.