棉花GhRAR1基因调控棉花抗黄萎病机理的研究

棉花黄萎病是一种严重影响棉株生长的土传维管束真菌病害。挖掘抗性基因,解析抗病机制,对创制棉花抗病种质防御棉花黄萎病具有重要意义。本研究从抗病棉花品种‘中植棉2号’中获得GhRAR1基因,采用病毒诱导的基因沉默(VIGS)技术、基因过表达及qRT-PCR技术探究GhRAR1介导的棉花抗黄萎病机制。结果表明,GhRAR1的表达受棉花黄萎病菌诱导。GhRAR1沉默棉株对黄萎病菌敏感性增强,表现为植株病情指数升高、叶片萎蔫、茎部维管束组织褐变加重。GhRAR1沉默棉株中活性氧(ROS)生成基因GhRbohD表达量低,H₂O₂积累量少。过表达GhRAR1基因的拟南芥植株对棉花黄萎病抗性增强,AtRbohD表达量高,H₂O₂积累量多,发生过敏反应(HR),菌丝扩散被抑制。推测GhRAR1可能调控ROS积累和HR,从而介导棉花对黄萎病菌的抗性。     

新疆主要棉区棉花黄萎病发生概况

为了解目前新疆棉花黄萎病的发生现状,对新疆主要植棉地区棉花黄萎病发生程度、落叶型菌系分布以及代表棉区主栽品种黄萎病抗性类型、棉花黄萎病发生规律进行了调查研究。结果表明:在调查的新疆棉田中棉花黄萎病发病田占58.2%,其中病情指数达5.0以上的棉田占28.1%;新疆棉花黄萎病菌系中37.3%为落叶型菌系;阿克苏棉区主栽的棉花品种(系)‘中棉49号’、‘中棉414号’、‘2905’棉花黄萎病抗性表现为耐病,生产上缺乏抗病品种。阿克苏棉区棉花黄萎病发生始期较往年趋早,发病程度趋于严重,6月下旬至7月下旬为棉黄萎病快速发展期,后期由于高温抑制作用,发病趋缓。     

几类抗虫棉品种对黄萎病抗性的调查

通过试验观察发现,目前推广的3类抗虫棉品种,虽在多年未种棉花的粮田种植,均发生了黄萎病,发病株率63.35%～81.65%,病情指数21.66～44.55;品种间存在着明显的差异.但杂交棉在抗黄萎病方面未表现出优势.因此,选用抗病性强的品种仍是棉花黄萎病综合防治的1项重要措施.     

棉花叶柄是分离黄萎病菌的最佳部位

棉花黄萎病是系统性土传病害。病菌从根部侵入棉株 ,随维管束扩展至棉株的各个部位。人们一般取茎基进行分离培养以获得黄萎菌 ,但我们在分离试验中发现 ,有时从茎基部难以分离出黄萎菌 ,尤其是枯、黄萎病混生棉株。为此 ,我们进行了棉株不同部位分离黄萎病菌的比较试验。经 4批共 2 9株的 1 51块茎基组织和 3 3 1块叶柄组织常规分离 ,在培养 6ｄ时 ,茎基和叶柄长出黄萎菌落的百分率平均分别为 3 9.7%和71 .0 %。结果表明 ,叶柄是分离黄萎菌的最佳部位。用叶柄分离的优点 :一是叶柄比茎基菌落出现早、多而纯。在ＰＳＡ、ＰＤＡ和水洋菜三种…     

不同机采棉种植模式对棉花主要植株形态影响效应分析

机采棉农艺与机艺高度配套是解决机采棉品质下降和降低植棉成本问题的关键。为了比较不同机采棉种植模式对棉花主要植株形态的影响效应,本研究以新陆中54号和新陆中75号为试验材料,采用目前大田条件下3种常用的机采棉种植模式,分别在花铃期、吐絮期对9个主要植株形态特征指标进行测定,研究机采棉种植模式植株形态指标间的相关性以及对其形态特征的影响。结果表明:不同机采棉种植模式对7个植株形态特征指标影响显著。相同机采棉种植模式下不同品种间始果节高度、始果节位和果枝长度等农艺性状差异显著。通过多重比较发现一膜四行较一膜六行机采棉种植模式棉花生育进程提前4～6 d,霜前花率提高2%～4%,单株结铃多1～2个。相关性分析结果表明机采种植模式下株高与始果节高度、果枝台数、叶形指数显著相关,相关系数分别为0.834、0.730、0.560,茎粗与单株结铃数呈显著正相关性,相关系数为0.735。因此,增加植株高度可使植株形态更符合机采要求,增加茎粗可使单株结铃数增加并且可减少倒伏,降低机采损失。植株过矮和过密已经无法适应机采棉种植要求,需打破传统的棉花"矮""密""早"高产栽培思维模式。     

棉花品种抗黄萎病性与体内生化成分相关分析

对棉花不同抗感黄萎病性品种生化成分分析结果表明:棉花品种在无菌土中体内可溶性还原糖含量相应地高于接菌土中的含量。蕾期抗耐品种平均含量为2.276％,高于感性品种的1.417％,而铃期趋势相反。叶片单宁含量高于根部。在无菌土中,抗耐品种叶片单宁含量平均为3.58％,高于感病品种的3.28％;而在接菌条件下趋势相反。根部棉酚含量显著高于叶部,但两者极显著相关。在无菌土和接菌土中,抗性品种棉酚含量均高于耐性品种,而耐性品种又高于感性品种。棉株体内棉酚含量与棉花品种抗黄萎病性显著正相关。同工酶谱测定显示,过氧化物酶在抗感品种的无病症叶片中,酶带特征基本一致,叶片显症后表现主酶带区加宽,主酶带数增多,变化趋势与症状严重度正相关。在显症条件下,抗性品种酯酶同工酶活性略强于感性品种;与无症叶相比,显症叶的SOD酶带数由原来的4—6条减少至3—4条,抗性品种的酶带数少于感性品种1—2条。     

内生球毛壳属真菌CEF-082对棉花黄萎病的控制作用

 内生真菌在生物防治上具有很大的应用潜力,本研究旨在明确棉花内生真菌球毛壳菌(Chaetomium globosum)CEF-082对黄萎病菌(Verticillium dahliae)的拮抗作用及对黄萎病(Verticillium wilt)的防治效果。采用圆盘滤膜法、凹玻片法等测定CEF-082对棉花黄萎病菌的抑制效果;温室采用滤液接种法和基质接种法测定其对棉花黄萎病的防治效果;并采用实时定量PCR检测棉株内防御基因的表达及黄萎病菌的含量。结果表明,CEF-082的非挥发性代谢物对棉花黄萎病菌菌落生长的抑制率为100%,对分生孢子产量的抑制率为70.5%,试验浓度范围内对分生孢子萌发没有明显抑制作用;棉苗预接种CEF-082培养滤液或将CEF-082的玉米沙粒培养物接入基质中再种植棉花,对黄萎病的防治效果分别为62.37%和66.88%;这两种处理对棉花的生长发育没有副作用,且后者对出苗有促进作用;荧光定量PCR结果表明,CEF-082代谢产物显著提高了棉花防御基因苯丙氨酸解氨酶基因(pal)、过氧化物酶基因(pod)和多酚氧化酶基因(ppo)以及β-1,3-glucanase基因的表达量,抑制了黄萎病菌在棉株体内的繁殖。棉花内生真菌CEF-082可以有效地防治棉花黄萎病,其作用机理主要有抗生作用和诱导抗性,在棉花黄萎病防治上具有较好的应用前景。     

转基因抗虫棉黄萎病综合防治技术体系

我国棉花生产已完成新一轮的品种更换,其主要特点是以抗虫棉代替常规棉。抗虫棉主要是美国33B及其衍生品种和国产GK系列品种,由于美国抗虫棉有别于我国现有品种的遗传基础,国产GK系列抗虫棉大部分具有南方品种背景,其病害种类和规律发生了一些变化。目前黄萎病已发展成为最主要的病害,控制该病的猖獗危害已成为棉花生产可持续发展的主要问题之一。为此,作者研究制定了一套"以抗(耐)病品种为基础,改善土壤生态条件及诱导棉株抗病性相结合控制棉花黄萎病危害"的转Bt基因抗虫棉黄萎病的综合防治技术体系,经过在北京、天津、河北、山东等地的生产应用,表明是一个有效的新型转Bt基因抗虫棉黄萎病IPM体系。     

4株拮抗细菌对棉花黄萎病的防治效果及机制

棉花黄萎病严重制约新疆棉花持续高产和稳产,拮抗细菌在棉花黄萎病生物防治中潜力巨大。本研究旨在明确4株拮抗细菌对棉花黄萎病的防治效果及其机制,为棉花黄萎病的生物防治提供理论依据。本文采用共培养法,研究4株拮抗细菌对黄萎病菌分生孢子浓度、菌丝和孢子形态、膜透性和产毒素能力的影响,采用滤液接种法进行盆栽试验验证其对棉花黄萎病的防治效果,采用愈创木酚法和氮蓝四唑光化还原法测定棉株体内防御酶系的活性。结果表明,4株拮抗细菌能够减少黄萎病菌分生孢子浓度,破坏其细胞形态,导致其细胞膜破裂,降低其毒蛋白浓度。接种4株拮抗细菌,能够诱导棉株体内POD酶和SOD酶等防御酶系的积累,提高了棉花的抗病能力,由菌株SHZ-24、SHT-15、SMT-24和BHZ-29处理的棉花,其对棉花黄萎病的最高防治效果分别为73.82%、80.48%、84.91%和84.18%。4株拮抗细菌通过抑制黄萎病菌的生长和诱导植物产生防御反应来提高对棉花黄萎病的抗性,其对棉花黄萎病具有良好的防治效果。     

新疆不同棉花品种游离脯氨酸与抗蚜性的关系

为了明确不同棉花品种游离脯氨酸含量与抗蚜性的关系,为早期鉴定棉花抗蚜性和抗性品种选育提供理论依据,对新疆棉区主栽的9个棉花品种进行了蕾期抗蚜性鉴定,测定了不同品种非受害与受蚜害条件下的游离脯氨酸含量.正常棉株与蚜害棉株蕾期游离脯氨酸含量均存在显著差异,高抗品种‘新海21号’和抗蚜品种‘新杂棉2号’的游离脯氨酸含量较高,棉株在受蚜虫刺吸胁迫后,游离脯氨酸含量大幅度增加,感蚜品种增加的幅度不如抗蚜品种明显,游离脯氨酸含量与蚜害指数呈极显著负相关.可以认为棉株中的游离脯氨酸是重要的抗蚜物质,在棉花品种固有抗蚜性及诱导抗蚜性中有明显作用.     

Influence of verticillium and fusarium wilts on the growth of melon

The susceptibility of the Greek melon cvs Kokkini Banana, Thraki, Peplos and Amynteou to Verticillium dahliae and Fusarium oxysporum f. sp. melonis was tested, by inoculating seedlings of the four cultivars by root immersion in a Verticillium and Fusarium inoculum suspension for 1 h. After 35 days, disease incidence was estimated from a disease index calculated as the product of the'leaf symptom index'and'vascular discoloration index'of each plant. In addition, plant height, main stem diameter, above-ground fresh and dry weight and root fresh and dry weight were measured. The disease severity of verticillium and fusarium wilts on plants was estimated by the correlation coefficient ( r ) between the disease index and the other characteristics. Both fungi had a significant negative effect on all the measured characteristics independently of the tested cultivar. Finally, the four melon cultivars showed different degrees of susceptibility to Verticillium and Fusarium indicating that tolerant selections can be achieved.     

Comparative effect of root-knot nematode on severity of verticillium and fusarium wilt in cotton

The effect of root-knot nematode (RKN) (Meloidogyne incognita) onVerticillium dahliae andFusarium oxysporum f.sp.vasinfectum in cotton (Gossypium hirsutum) was investigated. Two different inoculation methods were used, one in which inoculum was added to the soil, so that nematode and fungal inoculum were in close proximity; the other, inoculation into the stem, whereby the two inocula were spatially separated. Invasion of the roots by RKN enhanced disease severity, as measured by the height of vascular browning in the stem, following inoculation with either wilt pathogen. The effect of RKN on Fusarium wilt was more pronounced than that on Verticillium wilt. Nematode-enhanced infection byF. oxysporum is a well known effect but there are few reports of enhanced infection byVerticillium due to RKN. Relative resistance of a number of cotton cultivars to both wilt diseases, as measured by height of vascular browning, was similar to the known field performance of the cultivars. The use of vascular browning as an estimate of disease severity was therefore validated. http://www.phytoparasitica.org posting Feb. 3, 2003.     

Enhancement of germination of spores ofVerticillium dahliae andFusarium oxysporum f.sp.vasinfectum in vascular fluid from cotton plants infected with the root-knot nematode

Root-knot nematode [RKN] (Meloidogyne incognita) can increase the severity of Verticillium (V. dahliae) and Fusarium (F. oxysporum f.sp.vasinfectum) wilt diseases in cotton (Gossypium hirsutum). This study was conducted to determine some of the physiological responses caused by nematode invasion that might decrease resistance to vascular wilt diseases. The effect of RKN was investigated on spore germination and protein, carbohydrate and peroxidase content in the xylem fluids extracted from nematode-infected plants. Two cotton cultivars were used with different levels of resistance to both of the wilt pathogens. Spore germination was greater in the xylem fluids from nematode-infected plants than from nematode-free plants. The effect on spore germination was greater in theFusarium-resistant cultivar (51%). Analysis of these fluids showed a decrease in total protein and carbohydrate levels for both wilt-resistant cultivars, and an increase in peroxidase concentration. Fluids from nematode-free plants of theVerticillium-resistant cultivar contained 46% more peroxidase than theFusarium-resistant cultivar. The results provide further evidence that the effect of RKN on vascular wilt resistance is systemic and not only local. Changes in metabolites in the xylem pass from the root to the stem, accelerating disease development.     

Relationships Between Verticillium dahliae Inoculum Density and Wilt Incidence, Severity, and Growth of Cauliflower

ABSTRACT Microplot and field experiments were conducted to evaluate the effects of inoculum density on Verticillium wilt and cauliflower growth. Soil containing Verticillium dahliae microsclerotia was mixed with various proportions of fumigated soil to establish different inoculum densities (fumigated soil was used as the noninfested control). Seven inoculum density treatments replicated four times were established, and the treatments were arranged in a randomized complete block design. Soil was collected from each microplot immediately after soil infestation for V. dahliae assay by plating onto sodium polypectate agar (NP-10) selective medium using the Anderson sampler technique. Five-week-old cauliflower was transplanted into two beds within each 1.2- by 1.2-m microplot. At the same time, several extra plants were also transplanted at the edge of each bed for destructive sampling to examine the disease onset (vascular discoloration) after planting. Cauliflower plants were monitored for Verticillium wilt development. Stomatal resistance in two visually healthy upper and two lower, diseased leaves in each microplot was measured three times at weekly intervals after initial wilt symptoms occurred. At maturity, all plants were uprooted, washed free of soil, and wilt incidence and severity, plant height, number of leaves, and dry weights of leaves and roots were determined. The higher the inoculum density, the earlier was disease onset. A density of 4 microsclerotia per g of dry soil caused 16% wilt incidence, but about 10 microsclerotia per g of soil caused 50% wilt incidence. Both wilt incidence and severity increased with increasing inoculum density up to about 20 microsclerotia per g of soil, and additional inoculum did not result in significantly higher disease incidence and severity. A negative exponential model described the disease relationships to inoculum levels under both microplot and field conditions. Stomatal resistance of diseased leaves was significantly higher at higher inoculum densities; in healthy leaves, however, no treatment differences occurred. The height, number of leaves, and dry weights of leaves and roots of plants in the fumigated control were significantly higher than in infested treatments, but the effects of inoculum density treatments were variable between years. Timing of cauliflower infection, crop physiological processes related to hydraulic conductance, and wilt intensity (incidence and severity) were thus affected by the inoculum density. Verticillium wilt management methods used in cauliflower should reduce inoculum density to less than four micro-sclerotia per g of soil to produce crops with the fewest number of infected plants.     

新疆榆树黄萎病病原菌鉴定

2013年以来,在石河子大学农学院试验站榆树苗上,出现了一种病害,可导致叶片褪绿,变黄,萎蔫,甚至枯死,剖茎检查可见维管束变成褐色。采用常规组织分离法对病茎组织进行分离、纯化获得单孢纯培养菌株;通过常规纸钵撕底蘸根法对其进行致病性测定;用形态学和rDNA-ITS序列分析方法对病原菌进行鉴定。结果表明,分离菌株的菌落形态、分生孢子梗和分生孢子的形态都与大丽轮枝菌(Verticillium dahliae)一致;经分子生物学鉴定,该菌的rDNA-ITS序列与中国棉花黄萎病菌(V.dahliae)的ITS序列(登录号EU 835817.1)相似性达99.0%,故将引起新疆榆树黄萎病的病原菌鉴定为大丽轮枝菌(V.dahliae)。     

Dry mycelium of<Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> induces resistance against verticillium wilt and enhances growth of cotton plants

Dry mycelium (DM) ofPenicillium chrysogenum and its water extract (DME) were examined for their effects on induced resistance against Verticillium wilt and plant growth of cotton in the greenhouse. Soil application of 0.1–5% DM or 0.5–5% DME provided significant protection against the wilt, relative to the control. As neither DM nor DME inhibited mycelial growth ofVerticillium dahliae in vitro, it is suggested that the disease-controlling effects of DM or DME are attributed to induced resistance. DME (5%), as well as DME treated with chloroform or cold acetone, were as effective as 2% DM in reducing disease severity of Verticillium wilt, implying that the resistance-inducing substance(s) in DM are mostly water-soluble, with neither proteins nor lipids likely to be responsible for the induction of resistance. No significant difference in root colonization withV. dahliae was found between control-inoculated and 2% DM- or 5% DME-inoculated plants. However, colonization of hypocotyls and epicotyls was drastically suppressed by either 2% DM or 5% DME relative to the control. Treatments with 2% DM or 5% DME significantly increased ionically-bound peroxidase (POX) activity in roots, hypocotyls and the second leaf of cotton plants, with the hypocotyls expressing the highest increase. Soil application of DM or DME increased plant height, fresh and dry weight of inoculated and non-inoculated cotton plants, relative to their corresponding controls. It is concluded that DM may be used in cotton crops to promote plant growth and to induce resistance againstV. dahliae. POX might be associated with the defense against Verticillium wilt. http://www.phytoparasitica.org posting Jan. 9, 2002.     

Colonization in cotton plants by a green fluorescent protein labelled strain of Verticillium dahliae

Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease caused by the soil-borne fungal pathogen Verticillium dahliae. In this study, a green fluorescent protein (GFP) labelled V. dahliae strain (TV7) was obtained by transforming gfp into defoliating strain V991. Strain TV7 was used to study infection and colonization of wilt resistant cotton cultivar Zhongzhimian KV1 and susceptible cultivar 861 with the aid of confocal laser scanning microscopy. The results showed that initial infection and colonization of V. dahliae in Zhongzhimian KV1 and 861 were similar. Conidia and hyphal colonies formed and penetrated in the root meristematic and elongation zones and in the conjunction of the lateral and main roots. The invaded conidia started to germinate by 2 hpi (hours post-inoculation), penetrated into the root cortex and vascular bundles, eventually colonized in the stem xylem vessels and grew restrictedly in the individual tracheae of both resistant and susceptible cultivars. Moreover, pathogen DNA could be detected by qPCR in roots and stems of both cultivars, but its content in the wilt susceptible cultivar 861 was much higher than that in the wilt resistant cultivar Zhongzhimian KV1. The results indicated that the resistant cultivar has ability to suppress V. dahliae reproduction.     

Phytotoxicity on cotton ex-plants of an 18.5 kDa protein from culture filtrates of Verticillium dahliae

A phytotoxic protein that evokes the typical symptoms of Verticillium wilt disease in seedlings of Gossypium hirsutum L. (Upland cotton) was isolated from culture filtrates of Verticillium dahliae. The protein was purified by ammonium sulfate precipitation, Sephadex-G100 fractionation, and native PAGE. The 18.5 kDa protein, designated VD18.5, appears to be a single subunit protein with an isoelectric point between 3 and 5. VD18.5 induces symptoms of leaf dehydration, chlorosis, necrosis and stem discoloration in seedlings of the disease susceptible cotton cultivar Siokra 1–4. The LD₅₀ of VD18.5 on protoplasts of Siokra 1–4 was 18 μg mL⁻¹. VD18.5 had no noticeable effect on Pima S-7, which is a disease resistant cultivar. Phytotoxic activity was partially destroyed at high temperature and was abolished by digestion with proteinase K. Mass spectrometry fingerprinting and protein sequence data from VD18.5 yielded no significant matches when submitted to the Mascot search engine and NCBI non-redundant protein databases, respectively. These results suggest that VD18.5 is a novel protein that may be involved in the development of some of the symptoms associated with Verticillium wilt disease in the cotton plant.     

棉花黄萎病菌致病力测定及评价方法研究

 棉花黄萎病菌致病力测定及评价是抗病育种及病害综合防治的基础。本文以我国三大棉区的167个黄萎菌菌株为对象,研究致病力测定及评价中不同批次之间病情指数的校准及致病力类型划分标准等关键技术环节。结果表明,以中等致病力类型菌株Vd076为校正菌株,以感病性稳定的冀棉11为校正鉴别寄主,以校正菌株在校正鉴别寄主上病情指数达到50.0±5.0时的调查结果进行各菌株的致病力评价,可获得较好的校正效果,使不同批次试验数据具有可比性;依据聚类分析结果,制定了致病力类型划分标准,强、中、弱3种致病力类型的平均校正病指分别为﹥40.0、20.1~40.0和20.0;通过对不同鉴别寄主组合的分析,推荐陆地棉中棉所41号、豫棉21、鲁棉研28、中棉所35号、中棉所8号、冀棉11作为鉴别寄主。该研究为棉花黄萎病菌致病力变异等相关研究工作提供了技术支撑。     

Evaluation of Olive Cultivars for Resistance to Verticillium dahliae

Resistance of 23 important olive cultivars to Verticillium dahliae has been evaluated in four experiments under controlled conditions. Nine-month-old nursery olive plants were inoculated with a cotton non-defoliating (ND) (V4) or a cotton defoliating (D) (V117) isolate of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves. The percentage of plants killed and of those which recovered from the disease were used as additional parameters for including a particular cultivar into a defined category. Most of the evaluated cultivars were susceptible, although at different levels, to both isolates of V. dahliae. All cultivars were more susceptible to the D pathotype than to the ND one. A group of 11 cultivars, including several important Spanish cultivars, were susceptible or extremely susceptible to both pathotypes of V. dahliae. A second group showed differences of resistance depending on the pathotype used. They were susceptible or extremely susceptible to the D pathotype but resistant or moderately susceptible to the ND one. Finally, 'Frantoio', 'Oblonga' and 'Empeltre' were moderately susceptible to the D isolate of V. dahliae and resistant to the ND one. The resistance of 'Empeltre' was evident by the plant ability to recover from infection with either isolates. 'Empeltre' is considered to be a valuable cultivar for inclusion in breeding programmes for resistance to Verticillium wilt.