寡雄腐霉生防机理及应用研究进展

寡雄腐霉是一种重要的微生物农药,因其对病原菌具有广谱、高效的防治特性及对作物具有促生长和增产等特点被广泛关注。本文从寡雄腐霉对植物病原菌抗性、诱导植物产生诱导系统抗性、促进植物生长等方面对其生防的分子机理和信号转导的研究进展进行了综述,总结了寡雄腐霉在防治植物病害方面的应用,并对研究和应用过程中存在的问题及解决途径提出展望。     

寡雄腐霉发酵液对温室番茄生长及灰霉病的防治作用

为了研制高效、无毒的生物农药,采用自主分离的寡雄腐霉生防菌株Pythium oligandrum CQ2010制备发酵液,研究其对温室番茄生长及灰霉病的防治作用。结果表明,寡雄腐霉发酵液(Pythium oligandrum broth,POB)能显著提高番茄叶绿素含量,增强根系活力,增加氮、磷、钾吸收,促进植株生长,使生物量比对照提高65.14%;同时能激活与叶片抗病性相关的过氧化物酶和过氧化氢酶活性,诱导植株产生抗病性反应,提高抗病能力,其效果优于寡雄腐霉卵孢子制剂;而且POB能显著抑制离体灰葡萄孢菌的菌丝生长和孢子萌发;施用POB能使番茄灰霉病发病率下降22.28%~31.05%,病情指数从52.5(对照)下降至22.5~27.5,相对防治效果达47.62%~57.14%,叶片丙二醛含量显著降低,说明POB减轻了灰葡萄孢菌对细胞膜的伤害。表明POB兼具促进番茄生长及防治灰霉病的作用。     

寡雄腐霉重寄生作用的研究

 通过室内筛选和盆栽测定证明:寡雄腐霉(Pythium oligandrum)对Rhizoctoniasolanl AG-4,P.ultimum,P.spinosum和P.irregulare有抗生作用,其中以P.oligandr-um的卵孢子处理番茄种子对P.ultimum和P.solani AG-4引起的种腐,猝倒防治效果较好,防效分别为79.4%和63.6%。P.oligandrum对R.solani AG-4主要以菌寄生为主,菌丝能大量分枝侵入,而对P.ultimum主要以抗生为主,分泌出胞壁溶解物质溶解其菌丝。     

寡雄腐霉GAQ1对辣椒疫病的防效及对辣椒的促生作用

为明确自主分离的生防菌株寡雄腐霉Pythium oligandrum GAQ1对辣椒疫病的生防效果及其防御机制,通过平板拮抗和盆栽防效试验测定寡雄腐霉菌株GAQ1对辣椒疫霉Phytophthora capsici菌丝的拮抗作用、对辣椒疫病的防效和对辣椒的促生效果,同时应用实时荧光定量PCR技术检测菌株GAQ1处理后辣椒抗性基因表达的变化。结果表明,寡雄腐霉菌株GAQ1的菌丝可以缠绕并吸附寄生在辣椒疫霉菌丝表面或穿入菌丝体内,使辣椒疫霉菌丝细胞死亡;菌株GAQ1发酵液处理辣椒离体叶片再接种辣椒疫霉后产生的病斑直径较对照组显著减少,离体防效为30.79%;接种菌株GAQ1菌丝球后,辣椒疫病的病情指数较对照组显著降低,盆栽防效达69.16%;经菌株GAQ1处理辣椒后可诱导相关抗性基因PR1、WRKY40、WRKY53、ACCO和GST的相对表达量出现不同程度的升高,说明菌株GAQ1可诱导辣椒植株产生不同程度的防御系统应答。菌株GAQ1对辣椒具有良好的促生效果,处理后第5周其株高、株重及根重分别较对照组提高10.11%、33.23%和24.72%,其叶片中叶绿素a、叶绿素b及类胡萝卜素的含量分...     

寄生玉米的6种腐霉及其致病性研究

 从国内12省、自治区、直辖市玉米植株上分离出腐霉菌菌株,依据形态特征、培养性状、生长抑制温度,以及产生的有性与无性器官,鉴别出6个腐霉种:棘腐霉(Pythium acanthicum Drechsler),瓜果腐霉(P.aphanidermatum(Edson)Fitzpatrick),强雄腐霉(P. arrhenomanes Drechsler),禾生腐霉(P.graminicola Subramaniam),肿囊腐霉(P.inflatum Matthews),寡雄腐霉(P.oligandrum Drechsler),其中肿囊腐霉出现频率高,分布广泛。回接玉米表明,腐霉能够侵染玉米并引起病害,肿囊腐霉、禾生腐霉、强雄腐霉、棘腐霉是玉米青枯病的重要致病菌;瓜果腐霉能够引起玉米中部茎节腐烂;寡雄腐霉虽寄生玉米,但对玉米成株无致病力。     

寡雄腐霉与烯酰吗啉互作防治葡萄霜霉病和替代部分化学药剂减量用药应用

 利用室内与室外传统生物测定方法,结合诱导抗病性生理测定方法进行试验。明确寡雄腐霉用药时期;研发生化互作组合,明确增效作用机制;制定生化互作减量用药防控技术规程。结果表明寡雄腐霉预防葡萄霜霉病最佳时期为发病前1个月,连续喷雾用药3~4次,间隔期10~15 d。明确寡雄腐霉对叶片β-1,3葡聚糖酶产生具有诱导作用;研发出协同增效的生防菌剂与化学杀菌剂组合烯酰吗啉+寡雄腐霉(或者再加上100%硅氧烷化合物,此时同样防效下喷雾使用药液量仅为组合的40%)。寡雄腐霉与化学杀菌剂烯酰吗啉互作后,植株诱导抗病作用增强。制定以生防菌替代和高效合理化学用药为关键技术的减量用药防控葡萄霜霉病的综合体系,确定用药规程中药剂的使用类别、次序、排列和组合。     

寡雄腐霉发酵液的动物毒性及对辣椒的促生防病效应

为研制高效、无毒、低成本的生物农药以防治辣椒炭疽病,利用自主分离的寡雄腐霉Pythium oligandrum CQ2010菌株制备浓度为1.14‰发酵液,研究其对小鼠的急性毒性、对辣椒幼苗生长的影响及对辣椒炭疽病的防治效果。结果表明,小鼠1日内灌胃寡雄腐霉发酵液60 m L/kg,连续给药14 d后,对小鼠体重增长无显著影响,其外观和行为均无异常,组织器官也未见病理改变。寡雄腐霉发酵液可显著提高辣椒叶片叶绿素含量和根系活力,促进氮、磷、钾吸收,生物量比对照增加42.4%,效果优于寡雄腐霉卵孢子制剂;并能显著提高叶片中过氧化物酶活性,诱导植株抗病性。在辣椒接种炭疽病菌前后,寡雄腐霉发酵液均显著降低了叶片丙二醛的增幅,说明细胞膜受害减轻。施用寡雄腐霉发酵液可使辣椒炭疽病发病率和病情指数显著降低,分别为40.0%~46.7%和13.3~16.7,防治效果达59.9%~68.1%;在模拟自然发病试验中也能显著提高辣椒幼苗生物量,防治效果达55.4%。表明寡雄腐霉发酵液对动物安全无毒,能促进辣椒幼苗生长并防治炭疽病。     

寡雄腐霉发酵液对烤烟生长的影响及对烟草黑胫病的防治作用

为了探索对烟草黑胫病高效、稳定的生防制剂,利用寡雄腐霉工程菌株制备发酵液,采用温室盆栽试验研究该发酵液对烟苗生长的影响和对烟草黑胫病的防治作用。寡雄腐霉发酵液可提高正常烟苗生物量达22.6%,促进烟苗氮、磷、钾吸收;带病烟苗的发病率和病情指数分别降低了61.6%和64.2%,相对防治效果达64.2%;施用发酵液明显降低带病烟苗的细胞膜透性和丙二醛含量,提高烟叶多酚氧化酶、超氧化物歧化酶和苯丙氨酸解氨酶活性。说明寡雄腐霉发酵液可诱导烟株的防御作用,并提高其抗病能力和促生作用。     

木霉菌和腐霉菌对棉铃疫菌的作用研究

用玻璃纸对峙法观察到4种木霉(S-327,BT-12,BT-95,T-594)和寡雄腐霉(P.O.)对棉铃疫霉的营养生长均有一定抑制作用,但没有看到明显的抑菌圈。生物测定试验表明,拮抗菌不仅对棉铃疫霉有直接作用,而且还可以保护棉铃不受疫菌侵染。在5种拮抗菌中,P.O.和BT-12可能是防治棉铃疫病比较有潜力的生防菌。     

寡雄腐霉分泌物对植物病原真菌的抑制作用及其对番茄灰霉病的防治效果

为了探明寡雄腐霉分泌物中小分子量物质对病原真菌的作用,应用玻璃纸法和生长速率法测定了分子量小于8 kD的寡雄腐霉分泌物对9种植物病原真菌菌丝生长的抑制作用。结果表明,分子量小于8 kD的寡雄腐霉分泌物能强烈抑制森禾腐霉、灰葡萄孢、黄色镰刀菌的菌丝生长,抑菌率分别达到88．9%、86．7%和84．6%;对供试的4种腐霉以及尖孢镰刀菌的4个专化型的抑菌率均达显著性差异。在扫描电镜下可见,经寡雄腐霉分泌物处理的灰葡萄孢菌,菌丝细胞壁破裂、穿孔、干瘪,且菌丝分化出的分生孢子梗少,产孢量明显下降。温室盆栽试验表明,寡雄腐霉滤液对番茄灰霉病具有显著的预防保护作用与治疗作用,其预防保护作用与杀菌剂多菌灵1000倍的防治效果达到同一显著水平。     

Colonization of Pythium oligandrum in the tomato rhizosphere for biological control of bacterial wilt disease analyzed by real-time PCR and confocal laser-scanning microscopy

It recently has been reported that the non-plant-pathogenic oomycete Pythium oligandrum suppresses bacterial wilt caused by Ralstonia solanacearum in tomato. As one approach to determine disease-suppressive mechanisms of action, we analyzed the colonization of P. oligandrum in rhizospheres of tomato using real-time polymerase chain reaction (PCR) and confocal laser-scanning microscopy. The real-time PCR specifically quantified P. oligandrum in the tomato rhizosphere that is reliable over a range of 0.1 pg to 1 ng of P. oligandrum DNA from 25 mg dry weight of soil. Rhizosphere populations of P. oligandrum from tomato grown for 3 weeks in both unsterilized and sterilized field soils similarly increased with the initial application of at least 5 x 10(5) oospores per plant. Confocal microscopic observation also showed that hyphal development was frequent on the root surface and some hyphae penetrated into root epidermis. However, rhizosphere population dynamics after transplanting into sterilized soil showed that the P. oligandrum population decreased with time after transplanting, particularly at the root tips, indicating that this biocontrol fungus is rhizosphere competent but does not actively spread along roots. Protection over the long term from root-infecting pathogens does not seem to involve direct competition. However, sparse rhizosphere colonization of P. oligandrum reduced the bacterial wilt as well as more extensive colonization, which did not reduce the rhizosphere population of R. solanacearum. These results suggest that competition for infection sites and nutrients in rhizosphere is not the primary biocontrol mechanism of bacterial wilt by P. oligandrum.     

Treatment with the Mycoparasite Pythium oligandrum Triggers Induction of Defense-Related Reactions in Tomato Roots When Challenged with Fusarium oxysporum f. sp. radicis-lycopersici

ABSTRACT The influence exerted by the mycoparasite Pythium oligandrum in triggering plant defense reactions was investigated using an experimental system in which tomato plants were infected with the crown and root rot pathogen Fusarium oxysporum f. sp. radicis-lycopersici. To assess the antagonistic potential of P. oligandrum against F. oxysporum f. sp. radicis-lycopersici, the interaction between the two fungi was studied by scanning and transmission electron microscopy (SEM and TEM, respectively). SEM investigations of the interaction region between the fungi demonstrated that collapse and loss of turgor of F. oxysporum f. sp. radicis-lycopersici hyphae began soon after close contact was established with P. oligandrum. Ultrastructural observations confirmed that intimate contact between hyphae of P. oligandrum and cells of the pathogen resulted in a series of disturbances, including generalized disorganization of the host cytoplasm, retraction of the plasmalemma, and, finally, complete loss of the protoplasm. Cytochemical labeling of chitin with wheat germ agglutinin (WGA)/ovomucoid-gold complex showed that, except in the area of hyphal penetration, the chitin component of the host cell walls was structurally preserved at a time when the host cytoplasm had undergone complete disorganization. Interestingly, the same antagonistic process was observed in planta. The specific labeling patterns obtained with the exoglucanase-gold and WGA-ovomucoid-gold complexes confirmed that P. oligandrum successfully penetrated invading cells of the pathogen without causing substantial cell wall alterations, shown by the intense labeling of chitin. Cytological investigations of samples from P. oligandrum-inoculated tomato roots revealed that the fungus was able to colonize root tissues without inducing extensive cell damage. However, there was a novel finding concerning the structural alteration of the invading hyphae, evidenced by the frequent occurrence of empty fungal shells in root tissues. Pythium ingress in root tissues was associated with host metabolic changes, culminating in the elaboration of structural barriers at sites of potential fungal penetration. Striking differences in the extent of F. oxysporum f. sp. radicis-lycopersici colonization were observed between P. oligandrum-inoculated and control tomato plants. In control roots, the pathogen multiplied abundantly through much of the tissues, whereas in P. oligandrum-colonized roots pathogen growth was restricted to the outermost root tissues. This restricted pattern of pathogen colonization was accompanied by deposition of newly formed barriers beyond the infection sites. These host reactions appeared to be amplified compared to those seen in nonchallenged P. oligandrum-infected plants. Most hyphae of the pathogen that penetrated the epidermis exhibited considerable changes. Wall appositions contained large amounts of callose, in addition to be infiltrated with phenolic compounds. The labeling pattern obtained with gold-complexed laccase showed that phenolics were widely distributed in Fusarium-challenged P. oligandrum-inoculated tomato roots. Such compounds accumulated in the host cell walls and intercellular spaces. The wall-bound chitin component in Fusarium hyphae colonizing P. oligandrum-inoculated roots was preserved at a time when hyphae had undergone substantial degradation. These observations provide the first convincing evidence that P. oligandrum has the potential to induce plant defense reactions in addition to acting as a mycoparasite.     

Rhizosphere Competence of Pythium oligandrum

ABSTRACT The associations of Pythium oligandrum with the root cortex, rhizoplane, and rhizosphere were measured with 11 crop species. This work was expedited by the use of a semiselective technique for isolation of P. oligandrum from soil and plant material. Cortical colonization of roots by P. oligandrum was not detected, and the rhizoplanes of the roots of most crops were free of the fungus. However, P. oligandrum was detected in large quantities with every crop tested when roots with adhering soil (rhizosphere soil) were assayed. Different crop species and cultivars of cantaloupe, cauliflower, and tomato varied in rhizosphere densities of P. oligandrum, but rhizosphere population densities of the fungus were consistently higher than in nonrhizosphere soils with plants grown in P. oligandrum-infested sterilized potting mix or an unsterilized mineral soil. After transplanting tomatoes into potting mix infested with P. oligandrum, increases in CFU occurred over time in the rhizosphere but not in the nonrhizosphere soil. In trials on delivery methods of inoculum of P. oligandrum, the rhizosphere populations of tomato plants grown in potting mix were about sixfold higher compared to seed-coat treatments when ground, alginate pelleted oospores were applied to seedlings growing in plug containers prior to transplanting or to pots containing potting mix before direct seeding.     

Parasitism of macroconidia, chlamydospores and hyphae of Fusarium culmorum by mycoparasitic Pythium species

Parasitism of macroconidia and endoconidial chlamydospores of Fusarium culmorum by Pythium oligandrum was studied on water agar (WA), corn-meal agar (CMA) and glass slides. Loss of cytoplasmic content in F. culmorum spores was followed by complete degradation, and P. oligandrum produced an abundance of oogonia on the parasitized macroconidia. A simple method for assessing the relative aggressiveness of isolates is presented, based on the percentage of macroconidial cells devoid of cytoplasm. Parasitism of macroconidia by P. acanthophoron , P. oligandrum and P. periplocum , but not by the plant pathogenic species, P. tracheiphilum , was demonstrated by this method. Interactions between hyphae of P. oligandrum and F. culmorum on WA resulted in an increase in the number of oogonia of P. oligandrum and a decrease in the sporulation of F. culmorum . The ability of isolates of P. oligandrum , P. periplocum , P. acanthophoron and P. mycoparasiticum to suppress disease symptoms caused by F. culmorum on barley seedlings was demonstrated in a greenhouse test.     

Interactions Between the Mycoparasite Pythium oligandrum and Sclerotia of the Plant Pathogen Sclerotinia sclerotiorum

Pythium oligandrum Drechsler is a mycoparasite which parasitizes hyphae of many fungal species. A detailed study of the interactions between P. oligandrum and the sclerotia of the plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary is presented. Pythium oligandrum was present in Danish soils at concentrations between 4 and 26 cfu g^–1 soil. An increase in the natural population of P. oligandrum by addition of P. oligandrum zoospores to a soil reduced the ability of sclerotia of S. sclerotiorum to germinate myceliogenically and the sclerotia were colonized internally by P. oligandrum. This colonization and reduction of germination of sclerotia were also seen when sclerotia and P. oligandrum were incubated together in water. Small sclerotia were significantly more susceptible to parasitism by P. oligandrum than large sclerotia, and increasing the incubation time caused a further reduction in the germination ability of the sclerotia. P. oligandrum was able to pass through its entire life-cycle from zoospores to oogonia both with sclerotia as sole nutrient-source and in water containing exudates from the sclerotia. The cell wall degrading enzymes N-acetyl--D-glucosaminidase (NAGase), endo-chitinase, protease, -glucanase, -glucosidase and cellobiohydrolase were detected in culture filtrates of P. oligandrum cultivated with S. sclerotiorum. These findings suggest that P. oligandrum has a potential to reduce the survival of S. sclerotiorum sclerotia present naturally in soils, through mycoparasitic activity.     

Biological control of Pythium ultimum–induced damping-off by treating cress seed with the mycoparasite Pythium oligandrum

Incorporation of the aggressive mycoparasite Pythium oligandrum into a carboxymethyl cellulose-based seed coating decreased damping-off of cress seedlings by Pythium ultimum in both naturally infested soil and artificially infested sand. This effect is attributed to the germination of P. oligandrum oospores on the seed surface with subsequent generation of a protectant mycelium around the seedling.
P. oligandrum oospores survived storage periods of 10—20 weeks at zero relative humidity both on glass slides and within seed coatings. The mycoparasite also survived an 18–month burial in natural soil, probably in the form of oospores.
The implications of these observations for biocontrol of seedling diseases by P. oligandrum are discussed.     

Control of damping-off in cress and sugar-beet by commercial seed-coating with Pythium oligandrum

Seeds of cress and sugar-beet were coated with oospores of Pythium oligandrum using commercial seed-pelleting or film-coating procedures. Following either procedure approximately 10⁴ oospores were recovered from both seed types, achieving 75.94% of the targeted dose. Oospore germination (9.19%) was unaffected by the coating treatments. Both types of treatment reduced damping-off of cress caused by P. ultimum in artificially infested sand and potting compost and by Rhizoctonia solani in artificially infested sand. In some cases, the level of control was equivalent to fungicide drenches. In general, pelleting of P. oligandrum on cress gave better control than film-coating treatments. P. oligandrum also reduced damping-off of sugar-beet in soil naturally infested with Aphanomyces cochlioides and Pythium spp. Control was equivalent to that achieved with hymexazol fungicide seed-coating treatments and was related to the inoculum potential of A. cochlioides in the soil; neither standard hymexazol coatings nor P. oligandrum treatments gave control at high inoculum potentials. P. oligandrum was not rhizosphere competent on cress or sugar-beet.     

Mycoparasite-like behaviour of the plant pathogen Pythium aphanidermatum in vitro

Pythium aphanidermatum antagonized several other fungi after hyphal contact on films of water agar, recorded by video microscopy. It sometimes coiled round, penetrated and caused cytoplasmic coagulation of hyphae of the mycoparasites P. oligandrum, P. acanthophoron, P. mycoparasiticum and P. periplocum , but was not affected by them. It also coiled round, penetrated and coagulated P. ultimum, P. vexans, P. catenulatum, P. dissotocum and (infrequently) P. graminicola. It caused cytoplasmic coagulation of Fusarium oxysporum and Trichoderma aureoviride after contact, but without coiling. Six isolates of P. aphanidermatum behaved similarly, with a mode of action like that of the mycoparasite P. oligandrum but less rapid and consistent than this species. The results are discussed in relation to possible ecological roles of P. aphanidermatum.