Effect of plant roots on the germination of microsclerotia ofVerticillium dahliae

Induction of germination of microsclerotia by exudates from plant roots may be important for the control ofV. dahliae. Laboratory experiments with root observation boxes were carried out to assess the influence of root tips of seven crop species and cultivars on the germination of microsclerotia ofVerticillium dahliae in soil under controlled conditions. The root density of crops was measured in a field experiment. The results of the laboratory experiments and the field experiment were combined to estimate the total effect of crops on the population of microsclerotia in the field. Germination of microsclerotia was stimulated by all crops compared to a control without a crop. Among crops, roots of potato cvs Element and Astarte had a larger stimulation effect on microsclerotia than that of potato Ostara, pea, flax, sugar beet or onion. The number of hyphae per microsclerotium decreased with distance from the root surface regardless of the crop species or cultivar. Differences in root densities, in the affected root zones and in the stimulation effect on germination of microsclerotia caused large differences among crops in the effect on the population of microsclerotia in the soil. However, growing a rop with the special purpose to reduce the level ofV. dahliae inoculum in the soil is an inefficient control measure, because only a small part of the total soil volume is affected by roots and the number of hyphae per microscleroium affected is too low.Abbreviations MS microsclerotia, microsclerotium     

Application to soil of disinfectants through irrigation reduces Verticillium dahliae in the soil and verticillium wilt of olive

The application of disinfectants through drip irrigation could be a feasible practice against verticillium wilt (Verticillium dahliae) of olive. OX-VIRIN (activated peroxide) and OX-AGUA AL25 (quaternary ammonium compounds) are two disinfectants that have shown efficacy against V. dahliae in irrigation water and potential for reducing the disease in young olive plants. In this work, various post-planting application strategies incorporating OX-VIRIN (once a month, or twice a month on alternate or successive weeks) or OX-AGUA AL25 (once a month, or twice a month on alternate weeks) were assessed for their effect on V. dahliae in soil, disease in olive trees, and olive yield, in a 2-year pot-experiment under natural environmental conditions. The disinfectants were injected via metering pumps into a drip irrigation system that irrigated olive trees planted in V. dahliae-inoculated soil. All the application strategies significantly reduced the total inoculum density in soil compared to controls with no disinfectants and noninoculated soil. The microsclerotia density was also significantly reduced in disinfested soils by 73.6–86.8%, depending on the strategy. The symptoms and infection incidence were always lower in treatments subjected to disinfestation. The treatment with OX-AGUA AL25 applied twice a month on alternate weeks most reduced the symptoms (by 53.0%) and colonization index (by 70.8%) with respect to untreated water control. This soil disinfestation also significantly strengthened the symptom remission. Tree growth and production were negatively affected by soil inoculation (reduced by 45.6% and 88.7%, respectively), but not so by disinfectants, which even relieved the reduction in inoculated soils, especially when OX-AGUA AL25 was applied.     

一种快速检测向日葵地块土壤中黄萎病菌微菌核的新方法

由大丽轮枝孢菌侵染引起的向日葵黄萎病是一种重要的土传病害,微菌核是该病害主要的初侵染来源。目前,土壤中大丽轮枝孢菌微菌核的定量检测方法多操作步骤复杂繁琐,如利用PCR方法进行检测,对仪器设备和操作人员的素质都有较高的要求,而常规的土壤梯度稀释湿筛法的实验周期长且检测效率低,因此,建立一种快速定量检测土壤中大丽轮枝孢菌微菌核的方法,对于向日葵黄萎病的预报预测和防控非常重要。为了能够快速的定量检测土壤中微菌核的数量,以期探明不同耕作方式地块中土壤微菌核数量的差异,本实验建立了一套操作相对简单,实验周期较短的微菌核快速分离和定量检测的方法,即采样器—干筛法。该方法将微生物采样器和选择性培养基相结合,基于微生物采样器的撞击法原理,使土壤微生物粒子加速撞击到选择性培养基的培养皿表面,经培养后可见单菌落形成。利用该方法对内蒙古巴彦淖尔市不同的向日葵黄萎病发病地块中采集到的土壤样本中微菌核进行了定量检测,结果表明：两年向日葵连作地（样地1）土壤中微菌核的数量最多,平均每克土样中含有微菌核32.80个;与非寄主作物玉米轮作地块（样地2）土样中微菌核的数量最少,平均每克土样中含有微菌核11.80个,与寄主作物打籽葫芦轮作地块（样地3）微菌核数量介于二者之间。利用该方法能够明显区分不同地块土壤中微菌核的数量。通过和荧光定量PCR检测的结果进行相关性分析发现,该方法能够准确检测土壤中大丽轮枝孢菌微菌核的数量。     

蛋白激酶A催化亚基VdPKAC1对菌丝型大丽轮枝菌V07DF2培养性状及致病力的调控

【目的】通过同源重组敲除技术,解析菌丝型大丽轮枝菌中蛋白激酶A催化亚基基因VdPKAC1在调控微菌核发育、产孢及其致病力方面的作用。【方法】利用Invitrogen公司的Gateway®技术构建VdPKAC1的同源重组敲除质粒。该质粒的潮霉素抗性基因盒两端分别为靶标基因5′和3′端各1 kb的DNA序列。通过农杆菌AGL-1的介导,将该质粒的T-DNA区域整合到来源于棉花的菌丝型大丽轮枝菌V07DF2菌株中。从6个随机选择的含有潮霉素抗性基因片段的转化子中,通过靶标基因的PCR检测,获得了5个VdPKAC1基因敲除突变体。经过Southern杂交检测,选择了3个敲除突变体材料（2B5、C5和HH2）进行突变表型的观察和分析。在液体PDB培养7 d后观察敲除突变体与野生型菌株的黑色素产生情况;在固体PDA培养28 d后,观察敲除突变体与野生菌株的菌丝生长和休眠结构形成情况。此外,利用棉花根提取物对液体Cazpek-Dox培养7 d的各菌株分别进行诱导处理,在处理24、72 h后分别统计分生孢子数量,以此对敲除突变体和野生菌株的分生孢子产生能力进行评估。最后,将孢子浓度为1×107个/mL的突变体和野生菌株的分生孢子液灌根接种2叶期的棉花,测定其致病力。【结果】Southern杂交结果表明,在上述5个敲除突变体中,只有2B5和C5两个菌株为T-DNA单拷贝插入,而其他菌株均存在T-DNA异位整合的情况。在PDA培养条件下,3个敲除突变体（2B5、C5和HH2）与野生型相比具有黑色素合成增加,气生菌丝更加发达的特点。其中,敲除突变体2B5和C5在平板培养条件下还形成了野生型菌株没有的、与典型微菌核形态不同的深褐色“链状休眠菌丝体结构”。在棉花根提取物的诱导处理下,3个敲除突变体产生的分生孢子数量少于野生型菌株。另外,致病力分析结果表明,敲除突变体材料仍然具有一定的致病力,但却显著弱于野生菌株V07DF2。【结论】在大丽轮枝菌中,通过蛋白激酶A介导的环腺苷酸信号途径控制多种重要性状,而VdPKAC1是这一途径中的重要成员。以前的报道证实,VdPKAC1负调控微菌核的数量,而本研究利用菌丝型菌株V07DF2在PDA培养基上不产生微菌核的这一特性,通过同源重组原理敲除该负调控基因,成功获得可以在PDA上产生休眠结构的菌丝型大丽轮枝菌菌株的突变体。这些突变体材料将为大丽轮枝菌微菌核发育信号途径及其调控基因的发掘提供平台和基础。     

西兰花轮作及其残体还田对土壤中病原菌微菌核数量、黄萎病发生及马铃薯产量的影响

为明确西兰花轮作及其残体还田对土壤中病原菌大丽轮枝菌Verticillium dahliae微菌核数量、马铃薯黄萎病发生以及马铃薯产量的影响,分别于2017-2018年和2018-2019年在内蒙古自治区乌兰察布市凉城县岱海镇元山子农场人工病圃和锡林郭勒盟正蓝旗上都镇菜园村自然病圃开展田间试验,利用NP-10培养基测定不同处理土壤中的大丽轮枝菌微菌核数量,并对马铃薯地上叶片和地下块茎上黄萎病的发病率、病情指数以及马铃薯总产量和商品薯产量进行调查。结果显示,以马铃薯连作方式为对照,西兰花轮作及其残体还田后土壤中大丽轮枝菌微菌核的数量明显减少,其中在人工病圃于7、8和9月调查时分别降低了8.46%、36.41%和36.27%,在自然病圃于7、8和9月调查时则分别降低了7.22%、38.06%和48.84%;马铃薯地上叶片和地下块茎上黄萎病的发病率和病情指数均显著降低,在人工病圃对黄萎病的防效为32.91%和54.82%,在自然病圃对黄萎病的防效为46.72%和34.78%;马铃薯总产量和商品薯产量均有所提高,在人工病圃分别增产27.74%和40.08%,在自然病圃分别增产7.12%和8.77%。表明西兰花作为理想的轮作作物可应用于马铃薯黄萎病的有效防控。     

莱氏野村菌CQNr01微菌核的人工诱导培养

【目的】诱导莱氏野村菌的微菌核形成,为该菌靶标害虫的生物防治提供新的制剂类型和生产工艺。【方法】采用包含特定盐离子成分的液体培养基进行诱导培养,并对培养基中的碳﹑氮源浓度及碳氮比例进行优化;将微菌核干燥保存后复水测定菌核萌发率及对斜纹夜蛾幼虫的致病力。【结果】优化筛选出莱氏野村菌CQNr01微菌核诱导培养基,成功诱导莱氏野村菌菌株CQNr01产生细胞结构分化、色素沉着的微菌核结构;微菌核的产生数量受培养基中碳和氮素含量和相对比例的影响,在一定范围内,同样碳氮比情况下,低碳培养液有利于微菌核的产生。微菌核耐干燥保存,掺加硅藻土制备的微菌核制剂经干燥处理并贮存6个月吸水后仍能萌发产孢。微菌核制剂对斜纹夜蛾幼虫具有侵染活性,接种14 d后,供试昆虫的校正死亡率可达78.56%,供试浓度下,LT50约为7 d。【结论】莱氏野村菌的微菌核受特定培养基成分的诱导,获得的微菌核耐干燥且对寄主害虫具有很高的致死率,可以作为该菌生防制剂开发应用。     

棉田深翻对棉花黄萎病发病及其微菌核分布影响的初步研究

[目的]从棉花黄萎病微菌核含量水平揭示棉田深翻(深翻60 cm)对棉田棉花黄萎病发病的影响.[方法]利用MSM选择性培养基和水筛法分离土壤中的微菌核.[结果]深翻棉田土壤中的棉花黄萎菌微菌核数量低于常规棉田,深翻棉田棉花黄萎病发病程度也轻于常规棉田,说明深翻对棉花黄萎病的发生具有较明显的抑制作用.通过试验明确了棉田土壤中微菌核的数量,1年内有2次比较明显的消长,现蕾期出现第1个高峰,到吐絮期出现第2个高峰,微菌核数量越多病情越重.深翻田病情指数与微菌核数量的相关系数为0.891 1,常规棉田病情指数与微菌核数量的相关系数为0.450 3.[结论]棉田深翻能有效的降低棉花黄萎病的发生和危害.     

Patterns of endorhizal fungal associations in fruit crops of southern India

A survey on the endorhizal status of 39 fruit crops of 25 families, indicated that 22 fruit crops had arbuscular mycorrhizal (AM)–, four had dark septate endophyte (DSE)–fungal association, and 13 had dual colonization of AM and DSE fungi. Fruit crops were capable of forming Arum‐, Paris‐, or intermediate‐types of AM morphologies of which intermediate‐type was common. To our knowledge, we report for the first time AM in 10 fruit crops and DSE‐fungal association in 17 fruit crops. The extent of AM‐ and DSE‐fungal colonization ranged from 41% to 98% and < 1% to 89.9%, respectively, in different fruit crops. Arbuscular mycorrhizal–fungal spore numbers in the rhizosphere ranged from 6 to 61 spores per 25 g of soil. Arbuscular mycorrhizal–fungal spores belonging to Acaulospora, Glomus, and Scutellospora were isolated from the rhizosphere soil.     

土壤中黄萎菌核的含量对转基因棉花发病趋势的影响

利用土壤稀释法对转基因棉花材料12花龄期、吐絮期、收获期棉花根际土壤0～20 cm耕作层中黄萎菌核的含量和转基因材料7月31日至9月11日每隔10 d的发病情况进行了调查与分析.结果表明,转基因棉花材料12发病情况在7月31日至8月11日表现明显的高温隐症,病指下降,从8月11日至9月11日发病呈一直上升的趋势;每隔10 d转基因材料的平均病情指数分别为44.53;、42.53;、54.50;、61.17;和64.03;,整个调查发病期间呈较明显的双峰趋势.从该材料的根际土壤中分离出的黄萎病菌菌落呈黑色放射状,土壤中菌核含量在调查时期则呈先降低后上升又降低的趋势,调查的三个时期1 g土壤含微菌核量分别为118.80、202.80和105.20个/g.土壤中黄萎菌核的含量与该材料的发病情况有直接的关系,这主要与黄萎菌核的病原特性有关.     

大丽轮枝菌微菌核快速繁殖技术研究

[目的]筛选快速繁殖棉花黄萎病菌微菌核的适宜培养基.[方法]用6种培养基培养大丽轮枝菌(Verticillium dahliae Kleb.),通过生长速率、微菌核数量、微菌核直径和微菌核萌发率4项指标筛选合适的培养基,用5株大丽轮枝菌菌株对实验结果进行验证.[结果]改良燕麦培养基形成微菌核的数量和直径均优于其他培养基;M1和BMM培养基形成微菌核的数量较多,其微菌核直径均小于半组合培养基;验证的结果与实验结果基本一致.[结论]改良燕麦、M1和BMM培养基,根据实验需要均可作为快速繁殖微菌核的理想培养基.