Mechanism of Verticillium Wilt Stress Affecting Photosynthetic Characteristics and Chlorophyll Fluorescence Characteristics of Cotton Seedlings

[Objective] The goal of this experiment was to study the effects of Verticillium wilt on photosynthetic characteristics and chlorophyll fluorescence characteristics of cotton seedlings, and to provide a theoretical basis for the identification of cotton resistance to Verticillium wilt at the seedling stage and the establishment of Verticillium wilt control techniques. [Method] Zhongzhimian 2 (Verticillium wilt resistant cultivar, ZZM-2) and Jimian 11 (Verticillium wilt susceptible cultivar, JM-11) were selected. Verticillium dahliae was inoculated at the two true leaves stage. The photosynthetic and chlorophyll fluorescence parameters of the last fully expanded young leaves were measured at 6 d after inoculation. [Result] (1) Under the stress of Verticillium wilt, the leaf area per plant, leaf water potential, photosynthetic pigment content and net photosynthetic rate (Pn) of cotton seedlings decreased, and the decreases in ZZM-2 were smaller than those in JM-11. (2) The decreased intercellular CO2 concentration and increased stomatal limitation value in the two test materials indicated that the decrease in Pn was mainly caused by stomatal limitation, and the decrease in chlorophyll a in ZZM-2 was smaller than the decrease in chlorophyll b, whereas the opposite trend was observed in JM-11. (3) The light response curve parameters of ZZM-2 were increased and theCO₂ response curve parameters of ZZM-2 were decreased, but all parameters in JM-11 were decreased. For ZZM-2, the potential photochemical efficiency (F_v/F_m), the quantum yield of regulated energy dissipation (Y_NPQ) and the quantum yield of non-regulated energy dissipation (Y_NO) were increased slightly and the actual photochemical efficiency (Y_II) was decreased slightly, but the differences in these four indexes between diseased plants and healthy plants was not significant; however, the variable fluorescence yield (F_v) was significantly decreased. For JM-11, F_v, Fv/F_m, Y_II and Y_NPQ were significantly decreased, and Y_NO was significantly increased. (4) F_v/F_m in leaf lesion sites decreased, but that in non-lesion sites did not decline or even showed a slight increase. [Conclusion] Verticillium wilt resistant cultivars with higher photosynthetic capacity and carbon assimilation ability could reduce the effects of Verticillium wilt on photosynthetic characteristics and reduce the impacts on cotton yield and quality.     

Induction of Defense Enzymes and Control Effect of Endogenous and Rhizosphere Bacteria on Verticillium Wilt of Cotton

[Objective] The colonization in soil, promoting cotton growth effects, inducing resistance of cotton and controlling effects to cotton Verticillium wilt of Bacillus vanillea SMT-24, B. velezensis BHZ-29, B. subtilis SHT-15 and B. atrophaeus SHZ-24 were studied in this paper, which provides a scientific basis for better control of cotton Verticillium wilt. [Method] The antagonistic resistance of these bacteria to Verticillium dahliae was judged by observing the inhibition zone; the number of colonization in the rhizosphere soil was tested to represent the colonization ability of antagonistic bacteria; the growth promoting effect of strains on cotton was determined by analyzing cotton plant height, root length, root hair number and number of leaves; the effect of antagonistic strains on cotton induced resistance was explored by determining catalase (CAT), superoxide dismutase (SOD), phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) activities in cotton leaves, and investigating the disease index of cotton after inoculation with antagonistic bacteria solutions. [Result] SMT-24, BHZ-29, SHT-15 and SHZ-24 strains significantly inhibited the growth of V. dahliae and formed a clear zone of inhibition. After 2 to 22 days of the third inoculation, these strains of bacteria could survive in the root soil, increase the plant height, leaf number, and root hair number of cotton plants, as well as the activities of cotton CAT, SOD, PAL and POD enzymes, but reduced the disease index. [Conclusion] SMT-24, BHZ-29, SHT-15 and SHZ-24 strains can promote cotton growth, increase the activities of some related defense enzymes, and effectively control cotton Verticillium wilt.     

钾水平对小麦酚类物质、木质素代谢和接种蚜虫群体动态的影响

摘要：【目的】揭示钾素水平对小麦后期蚜虫抗性的影响效果及其机理, 为指导钾肥合理施用,降低小麦蚜虫危害提供依据。【方法】利用设置K0(0 )、K1(0.1 )、K2(0.15 )三个钾 (K2Omg/kg土)处理的盆栽试验并结合室内分析,研究了供钾水平对小麦后期酚类物质、木质素含量及其代谢相关酶活性的影响,比较了处理间接种蚜虫种群变化动态的差异。【结果】接虫后14-28d,单株小麦蚜虫数量不同施钾处理间出现明显的差异,其中以K0蚜虫群体最高,其次是K2,K1最低。群体高峰期出现时间K2、K0较K1提前2-3天。三个处理相比,K1植株总酚、酚酸、木质素含量及其代谢相关酶（PAL、TAL、POD、PPO）活性最高,其次是K2,K0最低。相关分析表明,植株总酚、酚酸、木质素含量及其代谢相关酶活性与植株钾素含量相关性不显著,但与植株干物质积累量及籽粒产量相关性显著。【结论】适量供钾可增加了植株对钾素的吸收,促进了植株生长和总酚、酚酸、木质素的合成及代谢相关酶的活性,从而可增加小麦对蚜虫的抗性;而供钾不足或过量,或降低了植株对钾素的吸收,或使植株对钾素产生奢移吸收,均不利于植株干物质积累和酚类物质、木质素的合成,从而降低了小麦对蚜虫的抗性。     

陆地棉植株组织结构和生化代谢与黄萎病抗性的关系

通过对8个陆地棉品种的植株组织结构、酶活性和根系分泌物的比较分析,研究陆地棉黄萎病抗性机制。研究结果表明,陆地棉抗病品种根和茎的导管细胞壁厚、直径小、数目多,髓射线数目多、单位面积薄壁细胞数多,有助于抵御棉花黄萎病菌的侵入与扩展。过氧化物酶（POD）活性与棉花抗黄萎病的关系不明显,但苯丙氨酸解氨酶（PAL     

Pathogenicity Differentiation and Pathotype Distribution of Verticillium dahliae from Cotton in Hunan Province

[Objective] The study of pathogenicity differentiation of Verticillium dahliae in the cotton growing areas in Hunan province could provide theories for breeding and promoting resistant varieties as well as integrated control of Verticillium wilt of cotton. [Method] Seventy seven strains of V. dahliae from major cotton growing areas in Hunan province were evaluated their cultural characteristics. The growth rate, sporulation quantity and pathogenicity of 31 strains among them were tested and SAS 9.1.3 statistical analysis software was used to cluster the pathogenicity of the tested strains. The special primer (D-1/D-2 and ND-1/ND-2) for polymerase chain reaction assay was used to test pathotype of 24 strains. [Result] According to their colony morphology on potato dextrose agar media and culture characteristics, the strains were classified into the sclerotium type, the hyphal type and the intermediate type accounted for 14.28%, 42.86% and 42.86%, respectively. The growth rate and sporulation quantity of the strains were 1.22–2.54 mm·d^－1 and 5.3×106–40.6×106 spore·mL^－1, which showed significant difference. In addition, based on the clustering results of average disease index,the strains were clustered into three groups which showed high (Type Ⅰ), low (Type Ⅱ) and moderate (Type Ⅲ) virulence to cotton with the proportion of 3.2%, 12.9% and 83.9%, respectively. [Conclusion] It is conclude that the main culture types of V. dahliae in Hunan province are the hyphal type and the intermediate type, and the strains showed pathogenicity differentiation. The result showed that defoliating pathotype of V. dahliae was dominant in the cotton growing areas of Hunan province．     

相对病情指数划分棉花品种抗病性的统计学基础

对多年棉花品种的黄萎病抗病性鉴定资料分析表明,同一个品种的黄萎病病情指数年度间差异变化较大。8年的鉴定资料统计结果表明,中棉所12的病情指数年度间变异系数为37.4％,冀棉11为33.3％,方差分析表明它们的病情指数年度间变异极显著。对1992—1997年中棉所12的病情指数进行概率统计,其总体平均数的估计值为62.0,95％置信区间为[58.4,65.6];99％置信区间为[57.2,66.8]。利用本文方法计算的相对病情指数来表示棉花品种的黄萎病感病性,与利用病情指数表示方法相比,可以将年度间变异降低60％,发生概率p=0.997。本方法划分的抗病性不同于目前通用的以一个年度田间病情指数划分的品种抗病性,而是概率保证下的抗病性,具有深层的数理统计的意义。     

Proteomics Analysis of Gossypium barbadense Leaves with Resistance to Verticillium dahliae under Infection Stress

[Objective] The purpose of this study is to analyze the resistance mechanism of island cotton under Verticillium wilt stress and to find possible resistance genes. [Method] The cotton resistance mechanism under the stress of Verticillium dahliae was studied by protein two-dimensional electrophoresis and mass spectrometry at the protein level. [Result] In the leaves, eleven proteins were down-regulated and 15 proteins were up-regulated after 2 hours of inoculation with Verticillium dahliae. The down-regulated proteins were mainly related to photosynthesis and carbon assimilation, and then we deduce that Verticillium dahliae is mainly broken cotton photosynthetic system to cause cotton susceptibility. The up-regulated proteins were mainly related to photosynthesis and benzoquinone reductase, beta-D-galactosidase, 14-3-3f protein and other disease-resistant protein. [Conclusion] It is speculated that the defense mechanism of island cotton on Verticillium wilt may occur at two levels: one is passive defense, it is manifested that the isoproteins of the down-regulated proteins such as chloroplast II AB binding protein and ribulose diphosphate carboxylase are highly expressed to maintain the stability of the island cotton photosynthetic system, the histone and 14-3-3f protein are highly expressed to keep the cell stability; the second is active defense, it is manifested that the high expressed β-D-galactosidase and benzoquinone reductase may be involved in the resistance of island cotton to Verticillium wilt.     

Isolation and Characterization of Endophytic Bacillus methylotrophicus LH-L3 Antagonist of Cotton Verticillium Wilt

The aim of this study is to obtain endophytic Bacillus with control effect on cotton Verticillium wilt. [Method] The cotton tissue grinding liquid was heated at 80 ℃, and spread onto the Luria-Bertani ager medium. The strains with better inhibitory effects on Verticillium dahliae Vd084, a strain with strong pathogenicity, was identified by the morphology, physiological and biochemical traits as well as 16S rRNA, gyrB and rpoB sequences, and its control effect on cotton Verticillium wilt was determined by pot experiments. [Result] 61 similar Bacillus strains were isolated from cotton plants. Among them, 17 strains could inhibit Verticillium dahliae Vd084. A strain named LH-L3 with a relatively higher antagonistic activity and a broad inhibition spectrum was obtained in the second screening. LH-L3 was classified into Bacillus methylotrophicus. Compared with the control, the emergence rate, plant height, root length, fresh weight of aerial part and underground part of cotton with 106 mL^－1 LH-L3 suspension drenching for 3 times increased by 42.85%, 10.24%, 23.83%, 10.05% and 97.62%, respectively. And control effect on Verticillium wilt was 85.24%. [Conclusion] LH-L3 had good growth promotion and Verticillium wilt control effects and showed good potential for biological control.     

拮抗细菌C-02防治棉花黄萎病的机理研究

 从抗生、竞争和诱导系统抗性三个方面对拮抗细菌C-02防治棉花黄萎病的机理展开了研究,结果表明,C-02分泌的抗菌蛋白能溶解病原菌VD-11的细胞壁,抑制其致萎毒素的分泌。与病原菌VD-11相比,C-02利用碳源有明显优势,在棉花根际土壤中具有显著的营养竞争优势和很好的定殖能力。C-02处理棉苗后,棉叶中植株防御性酶系PAL、POD和PPO的活性提高幅度大,维持时间长。C-02防治棉花黄萎病的机理是抗生、溶菌、竞争和诱导棉苗系统抗性的综合表现。     

The Plant Architecture of Direct-Sowing Cotton Planted after Barley Harvested with High Yield and Centralized Boll-Setting

[Object] The study was conducted to investigate the plant architecture characteristic of direct sowing cotton planted after barely harvested with high yield and centralized boll-setting in the Yangtze River basin. [Method] In 2014, the cultivars Guoxin 12-1, Yijimian and Lumianyan 36 were used and the two conventional fertilizer (CF) application rates (namely pure nitrogen 45 kg·hm^－2, 150 kg·hm^－2) and Guoxin 12-1 was used in 2015. Two slow release fertilizer (SR) utilization rates (namely pure nitrogen 150 kg·hm^－2 and 225 kg·hm^－2) and two SR topdressing at different growth stages (namely 100% topdressing at seedling stage, 70% topdressing at seedling stage + 30% at flowing stage) were set with CF (pure nitrogen 150 kg·hm^－2) and no fertilizer treatment as the controls. [Result] While the pure nitrogen (CF) amount was 150 kg·hm^－2, the seed cotton yield of Guoxin 12-1 were 4 014.72 kg·hm^－2. In 2015, the seed cotton yield for the treatment, application SR (pure nitrogen 150 kg·hm^－2)and application ratios of seedling stage and flowering stage of 100% and 0, respectively, increased by 30.96%. The ratios of bolls setting from 08-15 to 08-30 to total bolls (RBT) for the two treatments were 31.8% and 26.1%, respectively. Then a significantly positive correlation between the seed cotton yield and RBT was found（r2014＝0.948^**, r2015＝0.976^**）. Based on the analysis of relationship between the plant architecture indexes and RBT, plant architecture characteristics of cotton population with high yield and centralized boll-setting was proposed. [Conclusion] These indexes would be used to supervise the cotton culture management to achieve high yield and centralized boll-setting for the direct sowing cotton planted after barely harvested.     

棉花黄萎病抗性与过氧化物酶同工酶分析

采用等电聚焦电泳方法 ,对棉花二对抗黄萎病近等基因系以及三对色素腺体近等基因系的子叶进行过氧化物同工酶 (POD)分析。结果表明 ,色素腺体和棉酚含量与棉花抗性没有必然的联系 ;环境条件对棉花抗性表现有一定的影响 ,但对子叶中 POD同工酶谱带无显著的影响 ;4条 POD同工酶酶带 (P4、P6、P7、P10 )的表现与棉花抗黄萎病性有关 ,这些酶带的特殊表现可用于棉花抗病性辅助鉴定和辅助育种     

中国北方主要棉区土壤供钾能力及其与产量和品质的关系

2012―2013年在山东平原县、河北威县、新疆昌吉市代表性棉田上采集土壤(0~20 cm)和棉花样品,分析北方棉区土壤钾素状况、供钾能力及其与棉花产量和纤维品质的关系。结果表明,平原、威县、昌吉棉田速效钾含量平均分别为217、128、232 mg·kg-1,供钾能力平均分别为128.2、105.1、213.6 kg·hm-2,每生产100 kg皮棉棉花地上部分别吸收K 9.77、7.82、12.25 kg。不同棉区土壤速效钾含量与皮棉产量和地上部吸钾量相关性不同,在平原和昌吉,当土壤速效钾含量分别小于123 mg·kg-1和220 mg·kg-1时,与皮棉产量和地上部吸钾量具有显著正相关。3个植棉区棉花的吸钾量与子棉产量、皮棉产量都呈显著的正相关。棉花地上部吸钾量、纤维吸钾量和纤维含钾量与纤维品质指标的相关性不同地点差异大。     

Nitrogen Fertilizer and Its Residual Effect on Cotton Yield and Biomass Accumulation

[Objective] The optimum nitrogen rate of 300 kg·hm^－2 is well documented for cotton in the Yangtze River Valley, China. It can be reduced to 225 kg·hm^－2 without reducing yield in late sowing under high planting density. The aim of this study was to determine the possibility of further reducing the nitrogen application rate at the first flower stage, its residual effects and influence on cotton yield formation rule. [Method] A pot experiment was conducted with five nitrogen levels (120, 150, 180, 210 and 240 kg·hm^－2) in 2014, but only 180 kg·hm^－2(for studying nitrogen residual effect) in 2015, to study the cotton growth process, yield and its components and biomass accumulation. [Result] Nitrogen levels significantly affected the yield and biomass accumulation, but not the growth process and nitrogen residual effect. Maximum seed cotton yield (30.5 g·plant^－1), boll weight (3.8 g) and biomass accumulation, especially in the reproductive and vegetative organs, was recorded in the 180 kg·hm^－2 nitrogen treatment. In the rapid accumulation period, the proportion of biomass accumulation in the reproductive organs was the highest. [Conclusion] In a soil with medium fertility level, the application of 180 kg·hm^－2 nitrogen was optimal, because the strength of biomass accumulation in reproductive organs increased during the rapid accumulation period.     

黄瓜感染黑星病菌后的生理变化及抗病性的产生

对抗性不同黄瓜品种接种前后与抗性有关的酶活性、木质素含量、酚类物质含量进行测定，结果表明：接种前各品种的POD，PPO，PAL活性、木质素及酚类物质含量与品种的抗病性之间无相关性，接种后则与品种抗病性密切相关。抗病品种PAL活性峰在接种后24h出现，POD和PPO活性峰在接种后48～72h出现，酶活性提高幅度大；感病品种酶活性峰则在接种后72～96h出现，提高幅度小于抗病品种。抗病品种木质素含量的最大值出现在接种后24h，较未接种对照提高幅度大；感病品种木质素含量的最大值出现在接种后96h，较对照提高幅度小于抗病品种。接种后抗病品种比感病品种合成更多的酚类物质，而且酚类物质含量的变化与POD、PPO活性变化之间呈负相关性。     

Mapping of Verticillium Wilt Resistance QTL with Interspecific Advanced Backcross Population between Upland Cotton (Gossypium hirsutum L.) and Sea-Island Cotton (G. barbadense L.)

[Objective] The aim of this study was to map quantitative trait loci (QTLs) of Verticillium wilt resistance in cotton. [Method]In this study, with the cultivar Lumianyan 28(Gossypium hirsutumL.) as the recipient parent and the F₁generation obtained from the cross Hai7124 (G. barbadense) and TM-1(G. hirsutumL.) as the donor parent, an interspecific advanced backcross population was constructed. Compared with the integrated high density genetic linkage map published, simple sequence repeat(SSR) markers of polymorphism were used to construct genetic linkage map. QTLs detection was conducted by composite interval mapping method in field and disease nursery. [Result] 216 simple sequence repeated (SSR) markers of polymorphism were assigned to 26 Chromosomes with a total map distance of 3 380 cM and an average distance of about 15.77 cM between two adjacent markers. Six QTLs located on 6 chromosomes had been detected and could explain phenotypic variance with 8.56%～20.26%. Five of Six QTLs were consistent with the previous study. Moreover, one novel QTL which located on Chromosome 1 was detected in this study. These results maybe helpful for the marker-assisted development of new cultivars which were resistant to Verticillium wilt. [Conclusion] Six QTLs were detected, one of them is a new QTL on chromosome 1.     

基于人工病圃筛选和分子标记辅助的棉花抗黄萎病育种方法研究与应用

以抗病性较强的中植372为研究对象,和陆地棉感病品种军棉1号配制组合构建F2作图群体,筛选到和黄萎病抗性紧密连锁的SSR标记NAU1269。选育过程中,以中植372为父本或者母本,通过人工黄萎病病圃对种间杂交、回交、加代选育以及再杂交的材料进行了抗病性筛选,同时每代育种材料均对黄萎病抗性紧密连锁的SSR标记NAU1269进行跟踪检测,筛选出与黄萎病抗病性状紧密连锁的亲本及其后代材料,进而培育出抗黄萎病、产量高、品质优良的中植棉2号、新植5号、中植棉6号、中植棉8号等10余个国审及省审抗(耐)黄萎病棉花新品种。对育成的品种进行检测发现,中植2棉号、中植6棉号、中植8棉号和新植5号均能够检测到与黄萎病抗性紧密连锁的SSR标记NAU1269和已报道的抗黄萎病的分子标记NAU828和NAU1225,而且这3个标记在各个品种材料之间呈共显性分离。结果表明,基于人工病圃筛选和分子标记辅助育种相结合是选育棉花抗黄萎病材料可行、高效的育种方法。     

Identification of QTLs Related to Verticillium Wilt Resistance Based on RIL Population

[Objective] The objective of this study was to identify the stable quantitative trait loci (QTLs) related to Verticillium wilt resistance in cotton. [Method] In this study, a population of 111 recombinant inbred lines (RILs) strains was developed by crossing a highly resistant parental line "Changkangmian" to Verticillium wilt and the susceptible parent TM-1. The complete composite interval mapping method was adopted to detect QTLs by Verticillium wilt disease index in multiple environmental conditions and periods in Anyang and Verticillium wilt affected areas of Xinjiang. Simple sequence repeat (SSR) markers of polymorphism were screened for genetic mapping. [Result] The genetic map was constructed by 40 simple sequence repeat (SSR) markers, consisted of 12 linkage groups with total length of 212.5 centimorgan (cM). A total of six QTLs related to the resistance to Verticillium wilt were obtained. The likelihood of odd (LOD) values ranged from 2.51 to 5.55. The maximum phenotypic variation explained (PVE) 20.34%, and the minimum PVE 6.93% were recorded. Among detected QTLs, qVR-D05-1 was detected in both Verticillium wilt affected fields in Anyang and Xinjiang in July 2015 and July 2016 with PVE of 12.96% and 20.34%, respectively. [Conclusion] This study can provide a potential reference for mapping stable QTLs related to resistance to Verticillium wilt.