中国橡胶树疫霉种的研究*

疫病是我国植胶区的主要病害。近年来,作者从云南西双版纳和广东海南岛的橡胶树和胶园土共分离出57株疫霉菌种。通过分类研究,共鉴定出4个种:恶疫霉 Phytophthoracactorum(Leb.＆ Cohn)Schroeter,辣椒疫霉 P.capsici Leoman,柑桔褐腐疫霉 P.citrophthora(Sm.＆ Sm.)Leonian,和棕榈疫霉 P.palmivora(Butl.)Butler。其中辣椒疫霉是首次在橡胶树上发现。我国橡胶树疫霉的种群结构与东南亚和南亚的有所不同,除棕榈疫霉外,其余3种在东南亚和南亚均未发现。而东南亚常见种:簇囊疫霉(P.botryosa)、橡胶疫霉(P.heveae)和蜜色疫霉(P.meadii),在我国却迄今尚未发现或有待证实。以前报道分离自胶园土壤中的芋疫霉(P.colocasiae),可能系柑桔褐腐疫霉之误。绝大多数分离物经配对培养均可产生性器官:辣椒疫霉的A~1交配型和A~2交配型大致相等;柑桔褐腐疫霉和棕榈疫霉的A~2交配型则明显多于A~1交配型。     

福建、浙江、江苏、上海疫霉种的研究

1985—1986年,从福建漳州,浙江杭州,上海,江苏南京,苏州,扬州,常州等地采集100多种植物进行分离,在雪松(Cedrus deodara),倒挂仙人鞭(Cereus flagelliformis),冬青卫茅(Euonymus japonieus),构树(Broussonetia papyrifera),蓖麻(Ricinus communis),柑桔(Citrus reticulata),带叶兜兰(Paphiopedilum hirsutissimum),芋(Colocasia esculenta),鳄梨(Persea americana),山茶(Camellia sp.),珊瑚樱(Solanum pseudo-capsicum),百合(Lilium brownii var.viridulum),蔓常春花(Vinca major),西瓜(Citrullus lanatus)等14种寄主植物上分离到51个疫霉菌株,鉴定为9个种:烟草疫霉(=寄生疫霉)Phytophthora nicotianae B.de Haan(=P.parasitica Dast.),掘氏疫霉P.drechsleri Tucker,樟疫霉 P.ciunamomi Rands,棕榈疫霉P.palmivora(Butl.)Butl.,柑桔褐腐疫霉P.citrophthora(R.et E.Smith) Leon.,苎麻疫霉P.bochmeriae Saw.,蜜色疫霉P.meadii McRae,簇囊疫霉P.botryosa Chee,隐地疫霉P.cryptogea Pethyb.et Laff.。其中,烟草疫霉,掘氏疫霉,樟疫霉出现频率较高,分布较广,初步认为是上述地区植物疫病的主要病原菌。簇囊疫霉、蜜色疫霉是国内新记录。     

中国疫霉属异宗配合种的交配型

在疫霉属真菌中,很多种是重要的经济植物的病原菌,寄主范围广泛,包括乔木、灌木和各种农作物;为害性大,常带来严重的经济损失。本文主要研究疫霉异宗配合种的交配型。根据疫霉在纯培养和成对培养(dual culture)中产生有性器官的能力,疫霉属可分为同宗配合种和异宗配合种两大类群。异宗配合种的A~1交配型与同一种或其它种的A~2交配型进行成对培养时,可以形成有性器官。两个可亲合菌系配合而形成有性器官时,可能发生基因重组,其结果将使病原菌具有更强的生存能力、致病力以及更广泛的寄主范围。因此,研究疫霉两种不同的交配型的分布,不仅对认识病害的发生发展规律,进一步设计防治措施有着重要意义,而且对疫霉属的起源、演化和移栖也有着深远的理论意义。作者对收集到的7个异宗配合种:Phytophthora capsici,P.cinnamomi,P.citrophthora,P.colocasiae,P.infestans,P.nicotianae,P.palmivora的38个分离物进行了交配型的研究,测定工作使用澄清的Campbell蔬菜汁琼脂培养基(V8C),用于确定交配型的菌系有P.nicotianae var.parasitica A~1,P.nicotianae var.parasitica A~2,P.cinnamomi A~1,P.cinnamomi A~2,P.palmivora A~1,P.palmivora A~2.每个分离物分别与已知种的A~1和A~2两个菌系成对接种于同一V8C平板上,放入25℃温箱中培养,2周后在两个菌落的连线上检查有性器官的产生情况。实验结果表明,中国疫霉属异宗配合种的这些分离物的交配型与寄主或地理分布似无相关性,同一种植物上分离到的同种疫霉可以是A~1交配型,也可以为A~2交配型;同一地区可以出现两种交配型,不同地区又有相同的交配型。云南西双版纳橡胶园中的分离物(P.citrophthora,P.colocasiae,P.palmivora)都表现为中性。     

中国疫霉属真菌异宗配合的种的交配型

明确了异宗配合的疫霉属(Phytophthora)7个种(P.capsici,P.cinnamomi,P.drechsleri,P.melonis,P.nicotianae[=P.parasitica],P.palmivora 和 P.sinensis)47个菌株的交配型。特别有意义的是 P.drechsleri A~1和 A~2两个交配型在南京具有相同的优势,并且证实 P.cinnamomi A~1交配型的存在。     

掘氏疫霉同宗配合、异宗配合特性的研究

本文对掘氏疫霉的同宗配合、异宗配合特性进行了研究,发现新分离的掘氏疫霉A~2交配型菌株,不经过配对培养可以产生卵孢子;菌种在15℃下保存,自身可孕性能力逐渐减退。菌株通过接种黄瓜后,异宗配合特性可以恢复,同宗配合特性不能恢复。经过营养生长,无性繁殖,菌株后代的交配型不引起改变。通过对疫霉属有关种不同交配型与掘氏疫霉各菌株不同交配型配对培养,以及菌种不同保存时间、不同培养基、疫霉属有关种的种间和种内配对,讨论了掘氏疫霉同宗配合、异宗配合特性的影响因素。     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究*

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

虫霉目新种和新记录

本文报道了虫霉目的一个新种和3个新记录种。新种叶蝉虫疫霉(Erynia cicadellisLi et Fan sp.nov.)是我国南方稻叶蝉虫霉流行病的病原之一,与近缘种飞虱虫疫霉[E．Deiphaeis (Heri) Humber et Ben-Ze’ev] 的主要区别在于后者孢子较大,且不具有假根。蚜虫疫霉[E. aphidis (Hoffm.) Humber et Ben-Ze'ev]系世界不常见种,国内首次在陕西岚皋及福州的桃蚜上记录;飞虱虫疫霉和佩氏虫疫霉[E．Petchii Ben-Ze'ev et Kenneth)皆系南方褐稻虱虫霉流行病的病原。     

贵州地区木霉菌分离鉴定及对辣椒疫霉的拮抗作用

【背景】辣椒疫霉是一种毁灭性的土传病害,当前主要使用化学合成杀菌剂防治,但容易导致环境污染和食品安全等问题。【目的】筛选可拮抗辣椒疫霉的候选菌株,探究分离菌株拮抗辣椒疫霉的生理生化作用机制。【方法】综合应用形态学、核糖体RNA (rRNA)基因非转录区ITS序列相似性方法鉴定分离菌株,通过对峙实验筛选抑菌效果较高的拮抗菌株,基于比色法测定分离菌株发酵液粗提物对辣椒疫霉菌丝脂质过氧化、纤维素酶、β-葡萄糖苷酶(β-GC)和多聚半乳糖醛酸酶(PG)活性的影响。【结果】从腐木和土壤样品中分离得到11株木霉,分属于绿色木霉(Trichodermavirens)、哈茨木霉(Trichoderma harzianum)、钩状木霉(Trichoderma hamatum)和棘孢木霉(Trichoderma asperellum) 4个种。11株木霉对辣椒疫霉均有一定的抑制作用,抑制率达到90%以上的菌株包括:绿色木霉Tv-1(92.68%)、Tv-2 (95.12%),哈茨木霉Thz-2 (92.68%),钩状木霉Tha-1 (90.24%)。以4株高效木霉的发酵液粗提物处理辣椒疫霉菌丝5 d后,因脂质过氧化产生的丙二醛含量显著增加,分别达到1.20、1.48、2.69和3.16 nmol/g,显著高于对照处理的0.77 nmol/g;与对照组相比,β-GC、PG酶活性显著下降,分别降低了12.28%-64.91%、7.2%-15.5%;同时纤维素酶活性呈上升趋势,最显著组为2.647 U/mL,相对于对照组增加了0.831U/mL。【结论】分离得到4株明显抑制辣椒疫霉菌生长的高效木霉菌,主要通过破坏细胞壁结构、降低致病因子酶活力和增强脂质过氧化等方式起拮抗作用,可为辣椒疫病的生物防治提供理论依据和技术支持。     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究

本文对槐生疫霉、掘氏疫霉、樟疫霉、烟草疫霉、棕榈疫霉及辣椒疫霉等６种疫霉８１株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉     

Examination of some morphologically unusual cultures of Phytophthora species using a mitochondrial DNA miniprep technique and a standardised sporangium caducity assessment

Using the mitochondrial DNA miniprep technique, the identity of sixteen morphologically unusual cultures allocated to Phytophthora nicotianae, Phytophthora mexicana or Phytophthora porri was determined by comparison with a library of mtDNA band patterns obtained from reference cultures. Seven cultures were identified as Phytophthora nicotianae (including those assigned to Phytophthora mexicana and Phytophthora porri), six as strains of Phytophthora palmivora with small, ovoid, weakly caducous sporangia, and one as Phytophthora citrophthora. Some cultures of P. nicotianae had a low percentage of caducous sporangia. Percentage sporangium caducity, but not sporangium L : B ratio, is considered a useful taxonomic criterion for separating species morphologically similar to Phytophthora nicotianae. One culture from tobacco in New Zealand had a highly unusual morphology and a unique DNA band pattern, but was not identifiable. One culture from Acacia mearnsii in South Africa had a unique DNA band pattern which was identical to that of an isolate from Annona squamosa from Australia previously identified as Phytophthora palmivora, the precise identity of which is still unclear. The identity of most isolates from diseased durian was found to be Phytophthora palmivora, confirming its role as the main pathogen, but P. nicotianae was also identified from this host. This revised version was published online in August 2006 with corrections to the Cover Date.     

Variation Within and Between Phytophthora Species from Rubber and Citrus Trees in China, Determined by Polymerase Chain Reaction Using RAPDs

Variation among 39 isolates of Phytophthora of six morphological species (P. citrophthora. P. parasitka, P. capsici, P. palmivora and P. meadii. from rubber and citrus trees, and P. colocasiae from taro) was studied using random amplified polymorphic DNA (RAPD) analysis. Ten randomly-chosen 10-mer primers were used. Generally, the banding patterns were similar within species and different between species, but no one primer was able to distinguish all six species from one another. Cluster analysis on pooled data from all the primers gave six groups of isolates corresponding to the six morphological species. The group corresponding to P. citrophthora was divided further into subgroups that were related to host species and geographical location. This work confirmed the existing morphological classification of Phytophthora isolates from rubber and citrus trees in tropical China and showed the validity of using RAPDs to study the taxonomy of Phytophthora.     

A Survey of Phytophthora Species on Hainan Island of South China

During the period 1997–2007, a comprehensive study of the occurrence and distribution of Phytophthora species was conducted on Hainan Island of South China. To date, 14 species of Phytophthora have been recovered and their distribution determined. Phytophthora nicotianae ( =P. parasitica ) is the most important species attacking a wide variety of crops, followed by Phytophthora capsici and Phytophthora citrophthora . In contrast to Phytophthora colocasiae attacking taro leaves throughout the entire island, Phytophthora cyperi was found only once on Digitaria ciliaris in Danzhou. It is of interest to note that Phytophthora heveae, Phytophthora katsurae and Phytophthora insolita are commonly found in forest soil/water of protected mountains without causing any plant diseases. Although Phytophthora species are usually terrestrial or found in fresh water, one isolate of Phytophthora resembling closely the asexual isolates of P. insolita in Hainan was obtained from decaying Rhizophora leaves submerged in seawater. An unidentified Phytophthora species producing non-papillate; internally proliferating sporangia was isolated from the soil in which Ceriops tagel and Bruguiera serangula were growing in a salt water shrimp farm.     

有机酸在疫霉营养中作用的进一步研究

利用放射性同位素标记的~(14)C-琥珀酸跟踪,进一步证明有机酸在晚疫病菌的营养中作为碳架与铵盐结合而合成菌体的氨基酸,同时也进入三羧酸循环参加呼吸。所以其作用不仅仅是缓冲作用。所试6个生理小种19株国内外晚疫病菌都对有机酸有迫切需要而旺盛生长,只有两株国外菌种长势太弱。其他测试过的疫霉P.boehmeriae,P.capsici,P.cinnamomi,P.citrophthora,P.colocasiae,P.erythroseptica,P.fragariae,P.megasperma f.sp.glycinea,P.palmivora,P.parasitica,P.sinensis和P.syringae 12种包括A_1与A_2交配型共18株菌都对有机酸有需要,只在迫切程度上不同。但有机酸在疫霉属营养上有普遍意义则是显而易见的。     

Safflower Seedling a Selective Host to Discriminate Phytophthora melonis from Phytophthora drechsleri

Safflower seedlings were used to discriminate two morphologically similar species of Phytophthora namely P. melonis and P. drechsleri . All isolates of P. melonis from different sources could not infect safflower seedlings under high inoculum potential whereas all isolates of P. drechsleri from various hosts attacked safflower within a short period. Of 31 authentic Phytophthora species inoculated to safflower seedlings only seven species including Phytophthora asparagi , Phytophthora cactorum , Phytophthora cryptogea , Phytophthora drechsleri , Phytophthora erythroseptica , Phytophthora palmivora and Phytophthora quercina caused hypocotyls infection. All cucurbit isolates of Phytophthora from different parts of Iran and one from China could not infect safflower seedlings and were identified as P. melonis which had been confirmed previously by molecular analysis.     

Single-strand-conformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytophthora

Single-strand-conformation polymorphism (SSCP) of ribosomal DNA of 29 species (282 isolates) of Phytophthora was characterized in this study. Phytophthora boehmeriae, Phytophthora botryosa, Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora colocasiae, Phytophthora fragariae, Phytophthora heveae, Phytophthora hibernalis, Phytophthora ilicis, Phytophthora infestans, Phytophthora katsurae, Phytophthora lateralis, Phytophthora meadii, Phytophthora medicaginis, Phytophthora megakarya, Phytophthora nicotianae, Phytophthora palmivora, Phytophthora phaseoli, Phytophthora pseudotsugae, Phytophthora sojae, Phytophthora syringae, and Phytophthora tropicalis each showed a unique SSCP pattern. Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, and Phytophthora megasperma each had more than one distinct pattern. A single-stranded DNA ladder also was developed, which facilitates comparison of SSCP patterns within and between gels. With a single DNA fingerprint, 277 isolates of Phytophthora recovered from irrigation water and plant tissues in Virginia were all correctly identified into eight species at substantially reduced time, labor, and cost. The SSCP analysis presented in this work will aid in studies on taxonomy, genetics, and ecology of the genus Phytophthora.