Bacterial Antagonists of Aspergillus flavus

In order to search for bacteria capable of reducing the aflatoxin contamination of cottonseed, 892 indigenous bacterial isolates, including 11 that were endophytic to cotton, were screened for their ability to inhibit the growth of Aspergillus flavus on cottonseed in an in vitro bioassay. Only six isolates partially or totally inhibited fungal growth. All antagonistic isolates were recovered from boll, lint or seed surface or from the lint of mature bolls. One was retrieved from mature seeds. None of the endophytic isolates showed activity. In four field trials, the incidence of A. flavus -induced damage to locules inoculated simulteously with A. flavus plus the most A. flavus plus the most effective antagonistic isolate (D1) was reduced by 41-100% relative to locules inoculated with A. flavus alone. The severity of damage to locules inoculated simultaneously with A. flavus and with D1 was reduced by 60-l00% relative to locules inoculated with A. flavus alone. Isolate D1, identified as Pseudomonas cepacia, completely inhibited the growth of A. flavus on synthetic media.     

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.     

Expression of Pectinase Activity Among Aspergillus Flavus Isolates from Southwestern and Southeastern United States

Aspergillus flavus is a widely distributed filamentous fungus that contaminates crops with the potent carcinogen aflatoxin. This species can be divided into S and L strains on the basis of sclerotial morphology. During crop infection, A. flavus can secrete a large array of hydrolytic enzymes. These include pectinase, which aids fungal spread through plant tissues. A survey of pectinase expression by soil isolates derived from different regions of the United States revealed geographic polymorphisms. Strain L isolates from Arizona produced moderate to high levels of a specific pectinase P2c, while S strain isolates produced variable amounts of P2c. In contrast, L strain isolates from southeastern U.S. yielded variable P2c production, while S strain isolates consistently expressed high P2c levels. These results were corroborated by pectinase surveys of additional collections of A. flavus from soil and cottonseed. Expression patterns for P2c and pectinmethylesterase were evaluated for a select number of isolates using an isoelectric focusing technique. Clear zone reactions from the pectinase plate assay corresponded to the presence of P2c, while red ring reactions corresponded to the lack of P2c. Commercial cottonseed infected by S strain isolates frequently contained aflatoxin, even when infected by S strain isolates that did not produce pectinase P2c. Thus, although P2c-lacking isolates have reduced invasiveness, these isolates still have sufficient pathogenicity to cause aflatoxin contamination.     

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.     

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.     

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.     

The phylogenetics of mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called "S" and "L." The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as "S" or "L" based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the "S" sclerotium phenotype, whereas group I strains consisted of both "S" and "L" isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.     

Oviposition on and mining in bolls of Bt and non-Bt cotton by resistant and susceptible pink bollworm (Lepidoptera: Gelechiidae)

Transgenic cotton that produces insecticidal crystal protein Cry1Ac of Bacillus thuringiensis (Bt) has been effective in controlling pink bollworm, Pectinophora gossypiella (Saunders). We compared responses to bolls of Bt cotton and non-Bt cotton by adult females and neonates from susceptible and Cry1Ac-resistant strains of pink bollworm. In choice tests on caged cotton plants in the greenhouse, neither susceptible nor resistant females laid fewer eggs on Bt cotton bolls than on non-Bt cotton bolls, indicating that the Bt toxin did not deter oviposition. Multiple regression revealed that the number of eggs laid per boll was negatively associated with boll age and positively associated with boll diameter. Females also laid more eggs per boll on plants with more bolls. The distribution of eggs among bolls of Bt cotton and non-Bt cotton was clumped, indicating that boll quality rather than avoidance of previously laid eggs was a primary factor in oviposition preference. Parallel to the results from oviposition experiments, in laboratory no-choice tests with 10 neonates per boll, the number of entrance holes per boll did not differ between Bt cotton and non-Bt cotton for susceptible and resistant neonates. Also, like females, neonates preferred younger bolls and larger bolls. Thus, acceptance of bolls by females for oviposition and by neonates for mining was affected by boll age and diameter, but not by Bt toxin in bolls. The lack of discrimination between Bt and non-Bt cotton bolls by pink bollworm from susceptible and resistant strains indicates that oviposition and mining initiation are independent of susceptibility to Cry1Ac.     

The relative injuriousness of insect pests of cotton in the Namoi Valley,New South Wales

The sequence and relative injuriousness of insect pests was studied for three successive years in unsprayed cotton of the Namoi Valley. Heliothis punctigera Wallengren, the native budworm, and to a lesser extent H. armigera (Hübner), the cotton bollworm, prevented the setting of full crops by destroying buds and bolls. After setting, high proportions of bolls were injured by Earias huegeli Rogenhofer, the rough bollworm. Incidental damage was caused to seedlings by Thrips imaginis Bagnall, the plague thrips, and lo leaves by Anomis flava Fabricius, the cotton looper, and by Austracris guttulosa (Walker), the spur-throated locust. In commercial crops, pest control must rely on the use of broad-spectrum insecticides: H. armigera is currently resistant to several of these, and has become the principal species in the local population of cotton pests. A number of enviromental features impede the natural control of pests in the southerly areas where cotton is now produced. In comparison to the older dryland cropping practised in central Queensland, a higher yield is necessary to cover the production costs of irrigated cotton; a shorter growing season prevents the plants from compensating effectively for insect damage and the natural enemies of cotton pests are less abundant and less active.     

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 degrees 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 balls, 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.     

Stink Bug Feeding Induces Fluorescence in Developing Cotton Bolls

Background

Stink bugs (Hemiptera: Pentatomidae) comprise a critically important insect pest complex affecting 12 major crops worldwide including cotton. In the US, stink bug damage to developing cotton bolls causes boll abscission, lint staining, reduced fiber quality, and reduced yields with estimated losses ranging from 10 to 60 million dollars annually. Unfortunately, scouting for stink bug damage in the field is laborious and excessively time consuming. To improve scouting accuracy and efficiency, we investigated fluorescence changes in cotton boll tissues as a result of stink bug feeding.

Results

Fluorescent imaging under long-wave ultraviolet light showed that stink bug-damaged lint, the inner carpal wall, and the outside of the boll emitted strong blue-green fluorescence in a circular region near the puncture wound, whereas undamaged tissue emissions occurred at different wavelengths; the much weaker emission of undamaged tissue was dominated by chlorophyll fluorescence. We further characterized the optimum emission and excitation spectra to distinguish between stink bug damaged bolls from undamaged bolls.

Conclusions

The observed characteristic fluorescence peaks associated with stink bug damage give rise to a fluorescence-based method to rapidly distinguish between undamaged and stink bug damaged cotton bolls. Based on the fluorescent fingerprint, we envision a fluorescence reflectance imaging or a fluorescence ratiometric device to assist pest management professionals with rapidly determining the extent of stink bug damage in a cotton field.     

Transmission of cotton seed and boll rotting bacteria by the southern green stink bug (Nezara viridula L.)

AIMS: To determine the ability of the southern green stink bug (SGSB) (Nezara viridula L.) to transmit Pantoea agglomerans into cotton (Gossypium hirsutum) bolls. METHODS AND RESULTS: An SGSB laboratory colony was kept on fresh green beans. A P. agglomerans variant resistant to rifampicin (Rif) (strain Sc 1-R) was used as the opportunistic cotton pathogen. Adult insects were individually provided green beans that were sterilized and then soaked in either sterile water or in a suspension of strain Sc 1-R. Insects were individually caged with an unopened greenhouse-grown cotton boll. After 2 days, live SGSB were collected, surfaced sterilized, ground, serially diluted, and then plated on nonselective media and media amended with Rif. Exterior and interior evidence of feeding on bolls was recorded 2 weeks after exposure to insects. Seed and lint tissue were harvested, ground, serially diluted, and then plated on media with and without Rif. Bacteria were recovered on nonselective media from all insects, and from seed and lint with signs of insect feeding at concentrations ranging from 10(2) to 10(9) CFU g(-1) tissue. The Sc 1-R strain was isolated only from insects exposed to the marked strain and from seed and lint of respective bolls showing signs of insect feeding. Evidence of insect feeding on the exterior wall of the carpel was not always apparent (47%), whereas feeding was always observed (100%) on the interior wall in association with bacterial infections of seed and lint. CONCLUSIONS: Nezara viridula readily ingested the opportunistic P. agglomerans strain Sc 1-R and transmitted it into unopened cotton bolls. Infections by the transmitted Sc 1-R strain caused rotting of the entire locule that masked internal carpel wounds incurred by insect feeding. Bacteria were recovered from penetration points by insects not exposed to the pathogen, but locule damage was limited to the area surrounding the feeding site (c. 3 mm). SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that demonstrates the ability of SGSB to acquire and transmit plant pathogenic bacteria into cotton bolls.     

Effects of Growth-Inhibiting Substances from Young Cotton Bolls on Phosphorylation

The effects of growth-inhibiting substances from young cotton bolls on phosphorylation in both intact cotton plant and tissue slices of young cotton bolls were determined using 32P as a tracer. The results showed that the incorporation of 32P into the various organic phosphates including high-energy phosphate compounds, were decreased in abscissing bolls to about 1/2 as that of developing bolls. Infiltration of the growth-inhibiting substances into normal boll tissue caused a marked increase in respiration intensity, the synthesis of high-energy phosphate compouds was, however, decreased to 39% of that of the control tissue. When 10 ppm NAA was applied tvabscissing young cotton bolls, the incorporation of 32P into various fractions of organic phosphates increased, although never to the level of developing bolls. The above results suggested that uncoupling of respiration and oxidative phosphorylation might be one of the important effects of the growth-inhibiting substances in inducing the abscission of cotton bolls.     

Characterization of the damage of Spodoptera eridania (Cramer) and Spodoptera cosmioides (Walker) (Lepidoptera: Noctuidae) to structures of cotton plants

The cotton plant, Gossypium hirsutum, hosts various pests that damage different structures. Among these pests, Spodoptera cosmioides (Walker) and Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae) are considered important. The objectives of this study were to characterize and to quantify the potential damage of S. eridania and S. cosmioides feeding on different structures of cotton plants. For this purpose, newly-hatched larvae were reared on the following plant parts: leaf and flower bud; leaf and boll; flower bud or boll; and leaf, flower bud and boll. The survival of S. cosmioides and S. eridania was greater than 80% and 70% for larvae fed on cotton plant parts offered separately or together, respectively. One larva of S. eridania damaged 1.7 flower buds, but did not damage bolls, while one larva of S. cosmioides damaged 5.2 flower buds and 3.0 cotton bolls. Spodoptera eridania and S. cosmioides can be considered species with potential to cause economic damage to cotton plants because they can occur throughout cotton developmental stages causing defoliation and losses of reproductive structures. Therefore, the results validate field observations that these two species of Spodoptera are potential pests for cotton.     

Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

氮素对不同开花期棉铃纤维比强度形成的生理基础的影响

于2005年在江苏南京(长江流域下游棉区)和徐州(黄河流域黄淮棉区)棉田设置不同氮素水平(零氮：0 kg N·hm^-2,适氮：240 kg N·hm^-2,高氮：480 kg N·hm^-2)试验,研究氮素对不同开花期棉铃(伏前桃、伏桃和秋桃)纤维比强度形成生理基础的影响.结果表明：与适氮处理相比,零氮处理显著降低了棉铃对位叶氮浓度,增加了C/N,影响程度随开花期的推迟而加大,导致伏桃、秋桃对位叶制造和运输光合产物的能力在棉铃发育中后期大幅度下降,棉纤维的相对生长速率以及纤维发育关键酶蔗糖合成酶和β-1,3-葡聚糖酶活性降低,纤维素快速累积持续期缩短,纤维比强度显著降低;高氮处理显著增加了棉铃对位叶氮浓度,降低了C/N,影响程度随开花期的推迟而降低,其降低了伏前桃、伏桃发育过程中光合产物向纤维分配的比例、棉铃发育前中期的纤维发育关键酶活性及纤维素累积速率,导致其纤维比强度亦显著降低.综合分析认为,适宜的施氮量可以协调棉花的“源库”关系,有利于促进不同开花期棉铃高纤维比强度的形成.与适氮处理相比,零氮处理的伏前桃、伏桃和秋桃纤维比强度分别降低了1.8%、5.8%和13.0%,高氮处理则分别降低了8.2%、7.4%和-2.4%.     

江苏沿江棉区3代棉铃虫对棉花的为害及经济阈值

通过人工接虫为害,研究了3代棉铃虫对蕾铃脱落及结铃的影响,观察了不同年份棉铃虫卵和各龄幼虫的自然存活率及棉花蕾、幼铃的成铃率。结果表明,蕾铃的虫害脱落率对结铃的影响最大,直接通径系数达－0.8917,虫量和蕾铃脱落数主要通过接虫期间的虫害脱落率影响棉花结铃,其间接通径系数分别为－0.8894和-0.891,接虫期间的自然脱落率对结铃的直接和间接通径系数均很小,表明33代棉铃虫为害时棉花4的补偿能力已基本丧失,各龄幼虫的为害量观察表明,1－6龄幼虫的单虫为害当量分别为0.02、0.14、0.31、0.47、0.84和1.54个大铃,根据防治的直接和间接收益,得出3代棉铃虫的防治指标为百株累计卵量26粒。     

绿盲蝽对不同生长期棉花的刺吸危害特性

【目的】近年绿盲蝽Apolygus lucorum (Meyer-Dür)对棉花的危害成为农业中热议的话题和重点研究的课题。为更加深入了解此种害虫对棉花各组织的危害特性及量化危害,为早期预测预报提供依据,我们研究了绿盲蝽对棉花不同生长阶段（各组织）早期危害表征与后期成铃情况的关系。【方法】通过大田接虫法研究了绿盲蝽对苗期、蕾期、花期和铃期棉花植株的危害特性。【结果】绿盲蝽危害24 h会造成棉花子叶期和2叶期产生无头苗,2叶期后受绿盲蝽危害不会形成无头苗。建立了无头苗率和生长点被害级别的关系模型y=0.1906x-0.0439 (1≤x≤5)和棉花苗期（不同叶期）被害10 d后的叶片被害指数与刺吸点数和生长点被害级别回归模型：4叶期：y=0.532+0.202x₁+0.005x₂; 6叶期：y=-0.063+0.339x₁+0.002x₂; 8叶期：y=-0.150+0.087x₁+0.001x₂ (0≤x₁≤5, 1≤x₂≤5)。绿盲蝽对蕾和花的刺吸危害没有对后期棉铃的脱落率和大小产生显著影响。10 mm和15 mm直径棉铃被害后的校正脱落率分别为55.00%和26.60%,而直径大于15 mm的棉铃校正脱落率均为0;绿盲蝽的刺吸危害对棉铃的成铃大小无显著影响;建立了幼铃（10 mm、10~15 mm直径棉铃）刺吸点数和脱落率的回归模型［10 mm: y=0.1857x-0.081; 10~15 mm: y=0.0522x-0.0068（1≤x≤5）］。【结论】结果表明,幼苗阶段,棉花的子叶期和2叶期是受绿盲蝽危害的敏感时期;成株阶段,棉花嫩铃期是受绿盲蝽危害的敏感时期;建立的绿盲蝽刺吸危害的回归模型可用于绿盲蝽危害损失短期预测。因此,应在棉花受害敏感时期对绿盲蝽重点防治,提高防治效率。     

棉田三代玉米螟幼虫为害转移规律

1993年在江苏省如皋市研究了棉田三代玉米螟幼虫为害转移规律。结果表明,棉田三代玉米螟为害,因棉株生育状况而变,初龄幼虫为害表现为随机选择,在蕾、叶柄中的幼虫,2、3龄时向外转移为害。高龄幼虫绝大多数为害大铃,少量蛀杆。依据棉田发生为害规律,三代玉米螟防治时间,以卵盛期用药最佳。