Mycelia and spent fermentation broth of Beauveria bassiana incorporated into synthetic diets affect mortality,growth and development of larval Helicoverpa zea (Lepidoptera: Noctuidae)

Beauveria bassiana endophytically colonises corn (Zea mays) reducing tunneling from European corn borer (Ostrinia nubilalis). Endophytic colonisation of other plants by B. bassiana has been reported, and potentially, may reduce insect feeding on these plants. We evaluated the effects on larval growth and development, and mortality of different rates of dried, ground mycelia and water-soluble metabolites from fermentation broth culture of different isolates of B. bassiana incorporated into a synthetic diet and fed to neonate bollworm, Helicoverpa zea larvae. Development was delayed, weights of larvae were lower, and mortality was high for larvae fed the highest rates (1.0 and 5.0%, w/v) of mycelia incorporated diet compared to control. Insects fed diets containing mycelia of B. bassiana isolate 11-98 had the greatest mortality. Mortality was 100% for larvae fed 5% (w/v) mycelia incorporated diet of isolate 11-98, and 61% for isolate 3-00. For insects fed low rates (0.1 to 0.5%, w/v) of mycelia incorporated diet, mortality was lower, approximately 5% for isolate 11-98, and 5 to 14% for isolate 3-00. At the 0.1% (w/v) rate of mycelia incorporated diet, development occurred at an accelerated rate, compared to fungus-free controls, indicating increased nutrition in the lowest rate fungal diet. Mortality was low for all larvae fed diets containing spent fermentation broth of B. bassiana; however, development was delayed. Insects fed the highest rate (0.5%, v/v) of spent fermentation broth-amended diet had lower pupal weights, and a greater number of days to pupation than insects fed the lowest (0.1%, v/v) rate. Insects fed the 5% (v/v) rate of spent fermentation broth of isolates 11-98 and 3-00 had the longest days to pupation.     

The Entomopathogenic Fungal Endophytes Purpureocillium lilacinum (Formerly Paecilomyces lilacinus) and Beauveria bassiana Negatively Affect Cotton Aphid Reproduction under Both Greenhouse and Field Conditions

The effects of two entomopathogenic fungal endophytes, Beauveria bassiana and Purpureocillium lilacinum (formerly Paecilomyces lilacinus), were assessed on the reproduction of cotton aphid, Aphis gossypii Glover (Homoptera:Aphididae), through in planta feeding trials. In replicate greenhouse and field trials, cotton plants (Gossypium hirsutum) were inoculated as seed treatments with two concentrations of B. bassiana or P. lilacinum conidia. Positive colonization of cotton by the endophytes was confirmed through potato dextrose agar (PDA) media plating and PCR analysis. Inoculation and colonization of cotton by either B. bassiana or P. lilacinum negatively affected aphid reproduction over periods of seven and 14 days in a series of greenhouse trials. Field trials were conducted in the summers of 2012 and 2013 in which cotton plants inoculated as seed treatments with B. bassiana and P. lilacinum were exposed to cotton aphids for 14 days. There was a significant overall effect of endophyte treatment on the number of cotton aphids per plant. Plants inoculated with B. bassiana had significantly lower numbers of aphids across both years. The number of aphids on plants inoculated with P. lilacinum exhibited a similar, but non-significant, reduction in numbers relative to control plants. We also tested the pathogenicity of both P. lilacinum and B. bassiana strains used in the experiments against cotton aphids in a survival experiment where 60% and 57% of treated aphids, respectively, died from infection over seven days versus 10% mortality among control insects. Our results demonstrate (i) the successful establishment of P. lilacinum and B. bassiana as endophytes in cotton via seed inoculation, (ii) subsequent negative effects of the presence of both target endophytes on cotton aphid reproduction using whole plant assays, and (iii) that the P. lilacinum strain used is both endophytic and pathogenic to cotton aphids. Our results illustrate the potential of using these endophytes for the biological control of aphids and other herbivores under greenhouse and field conditions.     

Role of juvenile hormone esterase and epoxide hydrolase in reproduction of the cotton bollworm, Helicoverpa zea

The role of juvenile hormone (JH) esterase (JHE) and epoxide hydrolase (EH) in reproduction of the cotton bollworm, Helicoverpa zea, was investigated. Peak emergence of male and female bollworm adults occurred early in the scotophase. Female adults were added to males in a 1:2 ratio, respectively, at the beginning of the first photophase after emergence (d0). The highest oviposition rates for mated females were noted on d 2-4. The in vitro JH III esterase and JH III EH activity was measured in whole body homogenates of virgin and mated females from d0 to d8 post-emergence. Maximal JHE activity for virgin females occurred on d2 (1.09+/-0.14(+/-1 SEM) nmol of JH III degraded/min/mg protein), which was approximately twice that of mated females on the same day. The same results were observed for EH where the activity peaked on d2 at 0.053+/-0.003 as compared to 0.033+/-0.003 nmol of JH III degraded/min/mg protein, respectively. By d4, both JHE and JH EH activities declined significantly in virgin and mated females and were the same through d7. The developmental changes and effects of mating on JH degradation were similar when measured per insect. The highest levels of JHE and JH EH activity/min/mg protein in d2 virgin and mated females was found in ovaries followed by the carcass and then haemolymph; no EH activity was found in haemolymph as expected. For ovary, the JHE and JH EH activity was highest in virgin compared to mated females. The role of both enzymes in the regulation of reproduction is discussed.     

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.     

Inheritance of the fuzzless-lintless character in cotton (Gossypium hirsutum)

Summary A fuzzless-lintless mutant was identified in MCU.5 (Gossypium hirsutum) cotton in 1984. The inheritance of this character is reported in this paper. The fuzzless-lintless mutant was crossed with fuzzy-linted parents viz. MCU.5, MCU.7, Express Sindh (W), Piedmont Cleveland and Sindis Wild and the segregation pattern was studied in F₂ and BC₁F₁ generations. The segregation ratios for fuzzy-linted and fuzzless-lintless were 151 in the first cross, 631 in the second, third and fourth crosses and 2551 in the fifth cross. These ratios indicated that this character is controlled by 2–4 gene pairs, and the fuzzless-lintless character is a recessive to fuzzy-linted character. The chi-square test was significant only in the BC₁F₁ generation with MCU.7 and Express Sindh (W). The test revealed that the observed values deviated significantly from the expected ratio of 71, suggesting that this character is also influenced by modifier gene complex.     

Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum)

Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography–mass spectrometry (GC–MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.     

Characterization and expression analysis of TERMINAL FLOWER1 homologs from cultivated alloteraploid cotton (Gossypium hirsutum) and its diploid progenitors

The seasonal cycle and persistence of a plant is governed by a combination of the determinate or indeterminate status of shoot and root apical meristems. A perennial plant is one in which the apical meristem of at least one of its shoot axes remains indeterminate beyond the first growth season.TERMINAL FLOWER1 (TFL1) genes play important roles in regulating flowering time, the fate of inflorescence meristem and perenniality. To investigate the role of TFL1-like genes in the determination of the apical meristems in an industrially important crop cultivated for its fibers, we isolated and characterized two TFL1 homologs (TFL1a and TFL1b) from tetraploid cultivated cotton (Gossypium hirsutum) and its diploid progenitors (Gossypium arboreum and Gossypium raimondii). All isolated genes maintain the same exon–intron organization. Their phylogenetic analysis at the amino acid level confirmed that the isolated sequences are TFL1-like genes and collocate in the TFL1 clade of the PEBP protein family. Expression analysis revealed that the genes TFL1a and TFL1b have slightly different expression patterns, suggesting different functional roles in the determination of the meristems. Additionally, promoter analysis by computational methods revealed the presence of common binding motifs in TFL1-like promoters. These are the first reported TFL1-like genes isolated from cotton, the most important crop for the textile industry.     

Genetic mapping of a cross between Gossypium hirsutum (cotton) and the Hawaiian endemic, Gossypium tomentosum

The existence of five tetraploid species that derive from a common polyploidization event about 1 million years ago makes Gossypium (cotton) an attractive genus in which to study polyploid evolution and offers opportunities for crop improvement through introgression. To date, only crosses (HB) between the cultivated tetraploid cottons Gossypium hirsutum and G. barbadense have been genetically mapped. Genetic analysis of a cross (HT) between G. hirsutum and the Hawaiian endemic G. tomentosum is reported here. Overall, chromosomal lengths are closely correlated between the HB and HT maps, although there is generally more recombination in HT, consistent with a closer relationship between the two species. Interspecific differences in local recombination rates are observed, perhaps involving a number of possible factors. Our data corroborate cytogenetic evidence that chromosome arm translocations have not played a role in the divergence of polyploid cottons. However, one terminal inversion on chromosome (chr.) 3 does appear to differentiate G. tomentosum from G. barbadense; a few other apparent differences in marker order fall near gaps in the HT map and/or lack the suppression of recombination expected of inversions, and thus remain uncertain. Genetic analysis of a discrete trait that is characteristic of G. tomentosum, nectarilessness, mapped not to the classically reported location on chr. 12 but to the homoeologous location on chr. 26. We propose some hypotheses for further study to explore this incongruity. Preliminary quantitative trait locus (QTL) analysis of this small population, albeit with a high probability of false negatives, suggests a different genetic control of leaf morphology in HT than in HB, which also warrants further investigation.     

Medium components and culture conditions affect the thermotolerance of aerial conidia of fungal biocontrol agent Beauveria bassiana

AIMS: To produce more thermotolerable conidia of Beauveria bassiana, a well-known fungal biocontrol agent, by optimizing the medium components and culture conditions. METHODS AND RESULTS: The conidia produced on media including 0.5-6% glucose, sucrose or starch as carbon source and 50-300-microg ml(-1) Cu2+, Zn2+, Mn2+ or Fe3+ as additive to Sabouraud dextrose medium at 15-30 degrees C, pH 4-8 or KCl-adjusted water availabilities were exposed to 30-min wet heat stress at 48 degrees C. The medium components for conidial production with greatly enhanced thermotolerance included 4% glucose as optimum or 1% starch as alternative for the carbon source and < or =50-microg ml(-1) Mn2+ for the metal additive. The culture conditions were optimized as 25 degrees C and pH 5-6. Conidial thermotolerance decreased remarkably when sucrose and Fe3+ or Cu2+ were used in the cultures, but altered slightly when 50-200-microg ml(-1) Zn2+ were included. CONCLUSIONS: The tolerance of B. bassiana conidia to the thermal stress was significantly affected by the medium composition and culture conditions under which the conidia were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper treatment of small grains as mass production substrates for more glucose release and supplement of glucose or 50-microg ml(-1) Mn2+ are possible means to enhancing conidial thermotolerance and field persistence for improved insect control.     

NaCl胁迫对棉花种子萌发和幼苗生长的伤害

采用盐化土壤盆栽方法,选择耐盐性较强品种枝棉３号和耐盐性较弱品种泗棉２号,研究了盐分对棉花（Ｇｏｓｓｙｐｉｕｍ ｈｉｒｓｕｔｕｍ Ｌ．）种子萌发、出苗和幼苗生长的伤害。结果表明,在－０．５５和－１．１０ＭＰａ盐分胁迫下,对棉花伤害起决定性作用的因子是Ｎａ＾＋。２００ｍｍｏｌ／Ｌ ＮａＣｌ胁迫下,枝棉３号种子萌发时电导率上升幅度和子叶ＣＡＴ下降幅度均显著小于泗棉２号。１００和２００ｍｍｏｌ／Ｌ Ｎａ     

Characterization of the AlTI13 protein from Indian siris (Albizia lebbeck) that inhibits the growth of cotton bollworm (Helicoverpa armigera)

Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of AlTIs—1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel) were identified. The former was specific only toward trypsin. However, the latter was specific toward both trypsin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI₁₃) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH₄)₂SO₄ fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH₄)₂SO₄ F₃ fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%, respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a single dose of AlTI₁₃ (5 μg/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC₅₀) of AlTI₁₃ for trypsin and HaGPs were 0.14 and 0.17 μmol/ml, respectively. The optimum conditions for AlTI₁₃ were pH 8 and temperatures ranging from 35 to 40 °C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that ~ 28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that AlTIs, particularly, AlTI₁₃ of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.     

棉铃虫成虫种群空间结构的研究

利用性诱捕器调查了棉铃虫越冬代至 3代棉铃虫成虫种群的种群密度 ,而后用 4种地统计模型拟合其种群的空间结构 ,并分析它们的空间关系 ,其结果如下 :( 1 )越冬代种群在全方位、90°及 1 3 5°方向上空间模型均为线型 ;在 0°及 4 5°方向上分别为球型和指数型 ,模型的参数表明越冬代成虫的空间相关距离为 6.0 1～ 8.0 1空间单位 (单位为 1 0 0 m,下同 ) ,异质系数为 54.60～ 83 .50 ,空间依赖度为 1 50 .0 0～ 1 80 .60。 ( 2 )一代种群在全方位、0°、4 5°、90°及 1 3 5°方向上的空间模型分别为线型、球型、线型、球型和球型 ;模型的种群参数表明一代成虫的空间异质系数是 53 6.0 0～ 588.1 0 ,相关距离为1 6.0 2～ 1 9.0 7空间单位 ,空间取样方差约是 2 60 0。 ( 3 )二代成虫在全方位、0°和 4 5°方向的空间模型均为指数型 ,在 90°及1 3 5°方向的模型均为球型 ;模型的参数表明二代成虫的空间异质系数为 5.56～ 2 5.93 ,空间取样方差是 53 .4 9～ 64 .96,相关距离为 4 .54～ 5.3 3空间单位。 ( 4)三代种群在全方位、0°、4 5°、90°及 1 3 5°方向的模型分别为指数型、球型、指数型、线型和指数型 ,模型的参数表明三代成虫的空间异质系数为 2 91 .93～ 4 93 .67,空间取样方差为 1 1 99.53～ 2     

食物对棉铃虫生长发育及繁殖的影响

用棉花、花生、玉米及人工饲料作为食料,模拟幼虫田间取食习性,同时为雌蛾设立补充和无补充营养两个处理,研究了食物对棉铃虫 Helicoverpa armigera (Hübner) 生长发育及繁殖的影响。4种食料组幼虫历期之间有显著差异;雌、雄蛹重花生组显著小于其余三组。雌蛾腹部干重及其脂肪百分含量与雌蛹体重的排列顺序一样,雌蛾腹部干重人工饲料组显著大于棉花组和花生组。雌蛾寿命和繁殖受幼虫食料和成虫食物的双重影响。喂10%蜂蜜时,雌蛾寿命、交配率和产卵量在四个幼虫食料组之间没有显著差异。但喂以清水时,花生组雌蛾均未交配,寿命和产卵量显著小于其余三组。从同种幼虫食料来看,除人工饲料组外,其余3组雌蛾喂清水时的产卵量和寿命均比喂10%蜂蜜溶液时的显著下降。基于这些结果,作者认为棉花、玉米比花生更适合于棉铃虫的生长发育和繁殖。在田间自然栽培状态下,不同食料植物的糖分含量对棉铃虫生长发育和繁殖影响较大,含氮量的变化影响小。     

Changes in the level of tubulin subunits during development of cotton (Gossypium hirsutum) fiber

The levels of tubulin protein in developing cotton ( Gossypium hirsutum L. cv. Stoneville 825) fibers were measured from 8 to 28 days post-anthesis using commercially available monoclonal antibodies against alpha- and beta-tubulin. As the monoclonal antibodies against alpha- and beta-tubulin were prepared from yeast tubulin and chick brain tubulin, respectively, indirect immunofluorescence microscopy was used to establish that the two monoclonal antibodies recognized microtubule structures in cotton fibers. Western blots of electrophoretically separated proteins in crude extracts of cotton roots and fibers showed that single polypeptides with the expected apparent molecular weight for tubulin subunits were recognized by the antisera. An enzyme-linked immunosorbent assay was used to quantify tubulin levels. From 10 to 20 days post-anthesis the level of tubulin protein increases approximately three-fold. After 20 days post-anthesis, the amount of tubulin relative to total fiber protein reaches a plateau or decreases slightly. The rapid rise in tubulin is correlated with the elongation of the fiber and an increase in cellulose synthesis.     

The effects of root temperature and Ca supply on the growth and transpiration of cotton seedlings (Gossypium hirsutum,L.)

Summary Cotton seedlings were germinated in either tap water or a CaSO₄ solution and then grown for two days in nutrient solutions containing 0.1, 1.0, or 10.0 me Ca/1. They were then transferred to cultures having the same Ca variables and the roots subjected to temperatures of 26, 18, 15 and 12°C for four days. The fresh weight of all plant parts and leaf area increased with increasing root temperatures and with increasing Ca levels, the effect of Ca being most pronounced at the higher temperatures. The dry weight was increased by increasing root temperature; the effect of Ca was rather small. Water use increased with increasing temperature and was higher for the lowest Ca level than for the higher levels. The results emphasize the overriding effect of low temperature on water uptake. Calcium had little effect on the growth depression resulting from low root temperatures. Joint contribution from the Agronomy Department, Mississippi Agricultural Experiment Station, State College and the Soil Science Department, North Carolina Agricultural Experiment Station, Raleigh. Published with the approval of the Directors of Research as Journal Contribution No.1530 and Paper No.2389 of the Journal Series, respectively. This is a report of research conducted while the author was on sabbatical leave at North Carolina State University, February 1–September 10, 1965.     

棉花倍半萜合成酶基因GhTPS1的克隆及棉铃虫取食诱导表达谱分析

棉花被植食性昆虫取食后大量释放的特异性萜烯挥发物,能够有效吸引天敌昆虫进行寄主搜索和定位。本文从陆地棉(中棉12)Gossypium hirsutum叶片中克隆获得一个倍半萜合成酶基因的全长cDNA,命名为GhTPS1(GenBank登录号:JQ365627)。该基因编码545个氨基酸的蛋白,预测分子量为63.3 ku,等电点为5.92。氨基酸序列比对分析表明该基因与其它被子植物倍半萜合成酶基因一致性为34%～60%,其中与欧洲葡萄(-)-germacrene D synthase一致性最高(60%)。聚类分析表明GhTPS1属于由被子植物倍半萜合成酶基因组成的TPSa亚家族。采用实时荧光定量PCR检测了GhTPS1 mRNA在棉铃虫Helicoverpa armigera(Hübner)幼虫为害棉花不同时间的表达谱,结果表明接虫24 h该基因表达量显著上调。