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.     

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.     

Role of Pantoea agglomerans in opportunistic bacterial seed and boll rot of cotton (Gossypium hirsutum) grown in the field

AIMS: To investigate the aetiology of seed and boll rot of cotton grown in South Carolina (SC). METHODS AND RESULTS: Bacteria were isolated from diseased locules of cotton bolls collected in a field in SC, USA and tested for the ability to cause comparable disease symptoms in greenhouse grown cotton fruit. Spontaneously generated rifampicin-resistant (Rif(r)) mutants of the isolates were used in confirmatory pathogenicity tests. Resistance to the antibiotic was both stable and effective in differentiating between an inoculated Rif(r) strain, rifampicin-sensitive contaminants and/or endophytes. A series of inoculation methods was tested at various boll developmental stages and at different fruiting nodes on the plant. Field disease symptoms were reproduced by inoculating bolls at 2 weeks postanthesis with bacterial suspensions ranging from 10(3) to 10(6) CFU ml(-1). Pathogenic isolates were categorized as Pantoea agglomerans on the basis of phenotype testing, fatty acid profiling (similarity index = 0.94), and 16s ribosomal DNA sequence analysis (99% nucleotide identity). CONCLUSIONS: Pantoea agglomerans isolates from field-collected immature, diseased cotton caused comparable infection symptoms in greenhouse produced cotton fruit. SIGNIFICANCE AND IMPACT OF THE STUDY: In 1999, significant yield losses in SC cotton resulted from a previously unobserved seed and boll rot that has since been reported in other southeastern states. This study demonstrated a role of P. agglomerans in producing opportunistic bacterial seed and boll rot of cotton.     

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.     

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.     

Transfer of Alternaria macrospora from Cotton Seed to Seedling: Light and Scanning Electron Microscopy of Colonization

Alternaria macrospora is transferred from an infected seed, through the plant stem to the cotyledon and sporulates on its surface. However, at the beginning of the process, there is no difference between resistant and susceptible plants. In later stages, lower levels of fungal mycelium and sporulation were detected on the seedlings of a resistant cultivar. The fungus grew inside the plant from the underground to the upper parts. The mycelia which originated from surface spores could penetrate, through stomata and result in leaf colonization. A. macrospora sporulated ultimately on plant surfaces whereas the internal leaf tissue was colonized by mycelium only. This study suggests that cotyledon infection is a result of seed infection.     

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.     

Grass seed infection following inundation with Pyrenophora semeniperda

Seed infection by the seed-borne pathogen Pyrenophora semeniperda was demonstrated following inundative applications of the fungus to several annual grass weeds and wheat. The optimum time for applying inoculum was found to be around mid-anthesis in wheat. Applications of inoculum during and after anthesis in field experiments resulted in seed infection that manifested as stromatal development which affected germination or reduced seedling vigour. In an initial field experiment, >70% of Bromus diandrus seeds exhibited stromata of P. semeniperda. In a field comparison of inoculum types, conidial suspensions resulted in the greatest level of seed infection of B. diandrus compared with several types of inoculum containing mycelium fragments. Inundation of Avena fatua, Lolium rigidum, Hordeum leporinum, Vulpia bromoides and B. diandrus under field conditions with conidia of P. semeniperda also resulted in either the failure of infected seeds to germinate or a reduction of seedling vigour. It is concluded that the use of P. semeniperda as a seed-borne bioherbicide may be a biologically reasonable tactic, and the many logistical and technological constraints impeding its development are discussed.     

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.     

Potential for <Emphasis Type="Italic">Nezara</Emphasis><Emphasis Type="Italic">viridula</Emphasis> (Hemiptera: Pentatomidae) to Transmit Bacterial and Fungal Pathogens into Cotton Bolls

Recently, we showed that the southern green stink bug (SGSB), Nezara viridula (L.), can transmit Pantoea agglomerans (Ewing and Fife), an opportunistic bacterium, into green cotton bolls resulting in plant disease. Here, we hypothesized that our established model could be used to determine if the SGSB was a general, non-discriminate vector by using two other opportunistic bacterial pathogens of bolls (Pantoea ananatis [Serano] and Klebsiella pneumoniae [Schroeter]) and the known fungal pathogen Nematospora coryli (Peglion). Variants of P. ananatis (strain Pa-1R) and K. pneumoniae (strain Kp 5-1R) selected for rifampicin (Rif) resistance were used as bacterial opportunists. N. coryli was detected only from laboratory-reared SGSB directly exposed to the fungus. Both Pa-1R and Kp 5-1R were recovered from SGSB previously provided a contaminated food source (2 days), sterile food (5 days), and then harvested after being caged on bolls (2 days) at levels reaching 10³ and 10⁴ colony forming units (cfus) per insect, respectively. However, bolls caged with insects infected with Pa-1R or Kp 5-1R and with evidence of feeding did not become diseased nor were either opportunists detected from boll tissues. Insects infected with N. coryli transmitted the pathogen, which resulted in diseased bolls and fungi concentrations reached 10⁶ cfus/g locule tissue at 2 weeks following the caging period. Notably, each of the three pathogens independently caused boll disease when mechanically inoculated using a needle puncture. Generally, these results suggest that cotton pathogen acquisition by the SGSB was not sufficient to determine whether the insects were disease vectors of the opportunists.     

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.     

The effect of iprodione on the seed-borne phase of Alternaria brassicicola

Alternaria brassicicola infection of Brassica oleracea seeds was effectively controlled by a dust application of iprodione (Rovral 50% w.P.). At 2.5 g a.i./kg the seed-borne fungus was usually eliminated from samples with up to 61.5% affected seeds (35.5% internally diseased) but higher levels of infection required increased doses for complete eradication of the fungus. The germination of healthy seeds, including samples from 7–yr-old stocks, on filter paper was unaffected by the treatment. However, the germination of diseased samples, particularly those internally infected with A. brassicicola, was improved. More seedlings emerged from iprodione treated than from untreated seeds in glasshouse soil but the differences were not significant. The application of gamma-hexachlorocyclohexane to iprodione treated seeds sown in soil did not adversely affect subsequent emergence or disease control. Disease control was maintained and germination was not affected by the treatment when treated infected seeds were stored for 2 yr at 10 °C, 50% r.h. In a field trial iprodione seed treatment reduced seedling infection in a cabbage crop grown from naturally diseased seeds (100% contaminated, 45.5% internally infected) from 5.6 to 0.04%.     

Reducing boll weevil populations by clipping terminal buds and removing abscised fruiting bodies

Cotton pests damaging fruiting bodies (squares and young bolls) are difficult to control and their damage results in direct yield loss. Small growers, with low technological inputs, represent a large portion of cotton growers worldwide comprising more than 76 countries; they rely mainly on cultural practices to counteract pest attack in their crops. Boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), oviposition involves puncturing cotton squares and young bolls, causing abscission. We examined the impact on boll weevil population of collecting abscised cotton fruiting bodies and clipping plant terminals at 50% boll maturation in the field during two cotton‐growing seasons and under field cage conditions. Greatest numbers of damaged squares occurred ca. 117 days after planting and clipped plants resulted in reduction of abscised structures and adult boll weevils compared with non‐clipped plants, irrespective of cotton variety. Damaged young bolls were found ca. 128 days after planting in 2009 and 2011, but clipping had no effect. Numbers of boll weevils found in plants of the varieties BRS 201 and BRS Rubi (both in 2009) and BRS Rubi (in 2011) were, respectively, 13‐, 17‐, and 20‐fold greater when clipping plus collecting abscised fruiting bodies were not practiced. Furthermore, the average percentage of the boll weevil parasitoid Bracon vulgaris Ashmead (Hymenoptera: Braconidae) emerging from abscised and collected structures was similar between clipped and non‐clipped plant terminals in both seasons. Clipping plant terminals did not result in yield reduction and reduced adult boll weevil production. Collecting abscised reproductive structures, clipping plant terminals, and using both practices together reduced boll weevil populations by as much as 63, 57, and 79%, respectively, in cage trials. Thus, these practices cause significant impact on boll weevil populations and are feasible of adoption, especially for smallholder cotton growers.     

苗期遮荫对棉花产量与品质形成的影响

为揭示棉麦两熟共生期遮荫对棉花产量与品质形成的影响。在棉花苗期利用模拟棉麦两熟共生期遮荫的方法进行了研究。结果表明,遮荫对棉铃形成的影响因果枝,果节部位而异,遮荫有利于棉株下（1-3果枝）,中（4-6果枝）,上（7-9果枝）部果枝内围（1-2果节）铃的形成,对外围（≥3果节）尤其顶部果枝（≥10果枝）外围铃形成不利,从而决定铃重也随果枝,果节部位相应地变化,但遮荫对单株平均铃重的影响畔 小,变遮荫棉花籽棉产量而论,下,中部果枝的内围铃籽棉产量高于常规棉,在上,顶部果枝则相反,各部位果枝外围铃的籽棉产量均低于常规棉,遮荫棉花内,外围铃分布为1：0.36(常规棉为1：0.58),产量分布为1：0.42(常规棉为1：0.72)。苗期遮荫对棉纤维,棉籽品质性状的影响也主要在顶部果枝和上部果枝外围铃,综合分析遮荫棉花产量与品质的形成,棉花苗期耐遮荫性品种间存在差异。在本研究中以中9418耐遮荫性最强,中棉所19和春矮早次之。     

Longevity and egg development of adult female boll weevils fed exclusively on different parts and stages of cotton fruiting bodies

The capacities of the rind (i.e., the developing calyx and petals of squares; and the outer casing, or husk, of bolls) and the internal reproductive portion of cotton, Gossypium hirsutum L. (Malvaceae), squares of three sizes, and bolls of three ages to influence adult female boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), longevity and egg production were evaluated in laboratory bioassays. While feeding on the reproductive portion of squares was expected to support adult boll weevils for considerable periods of time (e.g., medium square reproductive portion: ≈185 days), feeding on rinds also resulted in substantial longevity (e.g., medium square rind: ≈120 days). As anticipated, feeding on the reproductive portion of squares resulted in formation of chorionated eggs in mated females, but a diet comprised exclusively of rinds, particularly from large squares, was associated with gravidity and high fecundity relative to rinds of young and mature bolls. The reproductive portion and rind of post‐bloom and young bolls as sources of food resulted in limited gravidity while mature bolls were not associated with any egg formation. These findings further our understanding of how boll weevils feed on different parts of the cotton plant and how those parts as food sources are related to the biology and ecology of the boll weevil.     

The Influence of the Inoculum Source of Rhizoctonia solani on Development of Black Scurf on Potato

Rhizoctonia solani, the causal agent of stem canker and black scurf on potato, survives as sclerotia on tubers, in soil and in plant residues. The objective of the present study was to evaluate the importance of inoculum source on disease development. Disease‐free minitubers and seed tubers contaminated with low levels of R. solani were planted in fumigated or artificially inoculated growth mixture in greenhouse experiments. Black scurf incidence and severity were significantly higher when the inoculum was present in both seed tubers and soil, compared with either of them separately. The severity of disease symptoms on the subterranean parts of the plant also were significantly higher in plots where both seed tubers and soil were contaminated, compared with plots where the inoculum source was either the seed tubers or the soil. Thus, both major sources of inoculum, seed tubers and soil, are important in disease development. However, when both sources are present, black scurf incidence and severity are increased, leading to economical damage to tuber yield and quality. Additional results from field trials support these findings. Disease incidence and severity on daughter tubers were correlated with levels of contamination in seed tubers and soil. When seed tubers and soil were heavily infested, the levels of black scurf incidence and severity on daughter tubers were very high; when seed tuber and soil infestation were very low, black scurf incidence and severity on progeny were also lower. Disease levels were reduced by in‐furrow fungicide treatment, but were less effective when the initial levels of the fungus on the seed tubers and in the soil were high.     

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     

Synchronicity of pollination and inoculation with Claviceps africana and its effects on pollen-pistil compatibility and seed production in sorghum

Sorghum ergot (caused by Claviceps africana) is a disease that affects sorghum seed development and yield. The interaction between pollen tube growth and hyphal development determines whether ovaries will be fertilized or colonized. Thus their respective deposition times on the stigma are critical. The effect of the time interval between pollination and inoculation on stigma receptivity and seed production was measured under field conditions in the male-sterile line A9 at Montecillo, State of México (2240m altitude). Pollination and inoculation treatments, from simultaneous application to 2 and 4h difference, were imposed when all stigmas on the panicle had emerged. Control panicles were either only pollinated or only inoculated. Eighteen hours later, pollen grains that adhered to, and germinated within the stigma, pollen tubes in the style and ovary, and fertilized pistils were counted. Pistils showing some disease expression (germinated spores, mycelium growth, or tissue necrosis) at 18, 48, and 72h were recorded. The number of diseased florets was registered at the dough growth stage, while number of seeds, grain yield and 100-seeds weight was measured at the physiological maturity. The pathogen applied in a water suspension of macro and secondary conidia caused a decrease in stigma receptivity; the greatest decrease (40-60%) occurred when the pollen and the inoculum were deposited almost simultaneously, regardless of which was deposited first. The route of the pollen tube was also the route for fungal infection. On average, treatments first inoculated had 60% more diseased florets and 36% less grain yield, 30% fewer seeds and seed size decreased 8%, than those first pollinated.     

Removal of early fruit branches triggered regulations in senescence,boll attributes and yield of Bt cotton genotypes

Premature senescence, unopened and rotted bolls are the primary constrictions hampering the accomplishment of yield potential in Bt cotton. An experiment was conducted with the objectives to delay senescence, enhance boll opening and reduce the boll rotting. The experiment was conducted at two randomly selected sites of University of Agriculture Faisalabad, Pakistan, during 2015 and repeated in 2016. Each experiment was laid out in randomised complete block design with two by two factorial arrangement structure and replicated five times. Treatments were comprised of R₀ = no fruit removal; R₁ = removal of early fruit branches (REFB) and two genotypes, viz. V₁ = FH‐142 and V₂ = MNH‐886. Genotypes were selected on the basis of abiotic stress tolerance (FH‐142) and susceptibility (MNH‐886). Nodes above cracked bolls, monopodial branches, opened bolls per plant and seed cotton yield were enhanced by 23, 12, 13 and 17%, respectively, under the influence of REFB over no fruit branch removal, while the REFB‐mediated decrease in boll rotting was 27%. Moreover, the response of MNH‐886 was more remarkable than FH‐142 for all studied variables. Conclusively, REFB increased the number of nodes above a cracked boll, monopodial branches, opened bolls per plant and seed cotton yield while decreased rotted bolls per plant in both genotypes.