丁香疫霉菌RPA/CRISPR-Cas12a快速检测方法的建立

丁香疫霉病菌（Phytophthora syringae,PS）造成数十种蔷薇科植物严重病害,是我国的植物检疫性有害生物。本研究根据GenBank中PS的Ras-like protein (Ypt1)基因,建立重组酶聚合酶等温扩增结合CRISPR-Cas12a系统的荧光法和侧向流层析试纸条快速检测方法,以实现在田间或口岸快速、准确、灵敏检测丁香疫霉病菌的目的。本研究优化了RPA/CRISPR-Cas12a的反应条件,考察了特异性、灵敏度以及实际样品检测能力。结果表明,该方法 37℃扩增40 min,能特异性地检测丁香疫霉菌,灵敏度为133 fg,与荧光定量PCR相当。本研究建立的快速检测方法可用于丁香疫霉菌的快速诊断。     

利用伏马毒素合成基因对玉米种子寄藏镰刀菌的分子检测

我国玉米种子普遍受到拟轮生镰刀菌(Fusarium verticillioides)的侵染,该菌产生的伏马毒素对国家种质库中玉米种质的安全保存有着潜在的影响。fum1基因是F.verticillioides等镰刀菌中伏马毒素生物合成的必需基因。选用3对已知引物Fum5F/Fum5R、P1/P2和P3/P4分别对从玉米种子中分离出的9株镰刀菌和1株阴性对照菌链格孢菌进行PCR扩增检测,在F001和F003菌株中扩增出相应的特异性表达片段。测序表明,这些片段的长度分别为846bp、888bp和703bp,与已知fum1基因(AF155773)的同源性达99%以上。3对引物的检测灵敏度达到88pg/mL。结果表明,这3对引物都能够有效地检测产生伏马毒素的镰刀菌菌株,但引物Fum5F/Fum5R所扩增片段完全来自fum1基因内部,具有更强的特异性。在此定性检测基础上,需要进一步研究定量检测方法并进一步研究伏马毒素对农作物种质保存的影响。     

冬生疫霉(Phytophthora hibernalis)的快速分子检测

 由冬生疫霉(Phytophthora hibernalis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了冬生疫霉和其它疫霉的ITS序列,在此基础上设计了一对检测冬生疫霉的特异性引物751F/752R,该对引物从冬生疫霉中扩增得到一条616bp的条带,而其它19种疫霉和其它真菌菌株均无扩增条带,表明该对引物对冬生疫霉具有特异性。在25μL PCR反应体系中,引物751F/752R检测灵敏度为10龟基因组DNA;而以卵菌ITS区通用引物ITS1/ITS4和751F/752R进行套式PCR扩增,能够检测到10ag的基因组DNA,使检测灵敏度提高了1000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子。结合快速碱裂解法提取发病组织的DNA,采用该PCR检测技术,在1个工作日内即可从人工接种发病的植物组织中特异性的检测到该病原菌。表明本研究建立的检测方法可用于冬生疫霉的快速分子检测。     

雪松疫霉(Phytophthora lateralis)的快速分子检测

由雪松疫霉(Phytophthora lateralis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了雪松疫霉和其他疫霉的tRNA序列,在此基础上设计了一对检测雪松疫霉的特异性引物T1/T2,该对引物从雪松疫霉中扩增得到1条192 bp的条带,而其他15种疫霉和其他真菌菌株均无扩增条带,表明该对引物对雪松疫霉具有特异性。在25μL PCR反应体系中,引物T1/T2检测灵敏度为10 pg基因组DNA;而以引物T3/T4和T1/T2进行巢式PCR扩增,能够检测到1 fg基因组DNA,使检测灵敏度提高了10 000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子,对人工接种发病的植物组织能够特异性地检测到该病原菌。此外,进一步建立了该病原菌的实时荧光定量PCR检测体系。     

马铃薯晚疫病菌对甲霜灵抗性的快速检测方法

为快速检测马铃薯晚疫病菌——致病疫霉Phytophthora infestans对甲霜灵的抗性,基于已知致病疫霉RPA190基因的T1145A变异引起的F382Y氨基酸点突变与甲霜灵抗性有关,通过设计4对特异性引物F382Y-F1/F382Y-R、F382Y-F2/F382Y-R、F382Y-F3/F382Y-R和F382Y-F4/F382Y-R建立等位基因特异性PCR(allele specific-polymerase chain reaction,AS-PCR)快速分子检测法,其中F382Y-R引物在4对引物里保持不变,并分析所建分子检测法的特异性和灵敏度。结果表明,正向特异性引物F382Y-F2、F382Y-F3和F382Y-F4的3''末端在致病疫霉抗甲霜灵菌株原有核苷酸突变位点1145A(对应引物为F382Y-F1)的基础上,再人工引入第1144位的核苷酸突变,将1144T分别突变成1144G、1144C和1144A,并优化退火温度和特异性引物与内参引物ITS1/ITS4比例,最终确定最适退火温度分别为54、60和58℃,特异性引物与内参引物最适浓度比例均为5∶1。以该3条引物对应的3对特异性引物和内参引物ITS1/ITS4组成的3组多重AS-PCR引物对甲霜灵敏感和抗性菌株具有良好的特异性,敏感菌株的扩增产物含1条879bp的内参片段,抗性菌株的扩增产物为1条879bp的内参片段和1条461bp的目的片段。3组AS-PCR检测体系均具有较高的灵敏度,其中引物F382Y-F4/F382Y-R对致病疫霉DNA的检测灵敏度达0.4pg/μL,引物F382Y-F2/F382Y-R和F382YF3/F382Y-R的检测灵敏度达4pg/μL。     

基于环介导等温扩增技术检测由强雄腐霉引起的玉米茎腐病

为建立强雄腐霉Pythium arrhenomanes的快速分子检测技术,基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)以β-tubulin基因为靶标序列,设计强雄腐霉的特异性引物,建立了一种准确快速的LAMP检测方法,并对该检测方法的特异性、灵敏度和实际应用效果进行评估。25μL LAMP最终反应体系为:10×ThermoPol Buffer 2.5μL、10 mmol/L dNTPs 3.5μL、6 mmol/L MgSO_4 2μL、5 mol/L甜菜碱4μL、40μmol/L FIP-1/BIP-1各1μL、5μmol/L F3-1和B3-1各1μL、40μmol/L LB-1 1μL、Bst DNA polymerase 1μL、2.5 mmol/L HNB 1.9μL、模板DNA 2μL、灭菌水3.1μL。LAMP体系在等温64℃条件下反应60 min,HNB显色反应显示天蓝色即为阳性反应;扩增产物经2%琼脂糖凝胶电泳验证为梯形条带,也可判定为阳性反应。特异性检测结果显示,LAMP体系能特异性地检测出强雄腐霉,而其它卵菌近缘种和常见植物病原真菌均未检测出。灵敏度检测结果显示,LAMP体系的最低检测灵敏度为10 pg/μL,是普通PCR反应灵敏度的1 000倍。在实际应用中,LAMP体系能够快速检测出人工接种和实际发病玉米组织中的病原菌。表明本研究建立的快速、准确、可视化的LAMP检测方法能在60 min内检测出强雄腐霉。     

黄花菜叶斑病菌鉴定、生物学特性分析、防治药剂筛选及LAMP检测体系的建立

为明确浙江省黄花菜叶斑病病原菌的种类，探究其对杀菌剂的敏感性并建立快捷检测方法，采用组织分离法获得黄花菜叶斑病菌，通过形态学特征观察和分子生物学技术对病原菌进行鉴定，测定其致病力及生物学特性，分析6种杀菌剂对病原菌的生长抑制效果，并以ITS和EF-1α序列为靶标设计特异性引物建立环介导等温扩增（loop-mediated isothermal amplification，LAMP）检测方法。结果表明，根据形态学特征以及ITS与EF-1α序列分析结果确定黄花菜叶斑病菌为杏黄镰孢Fusarium armeniacum。该病原菌菌丝生长适宜温度为20~30℃，pH 7时菌丝生长最快，以葡萄糖为碳源、硝酸钠为氮源时利用率最高。96%己唑醇对菌丝生长的抑制作用最强，EC₅₀为0.119 μg/mL。本研究所建立的LAMP体系在60℃条件下反应60 min，琼脂糖凝胶电泳和SYBR Green I染料染色均可判断检测结果。该方法可特异性地检测出杏黄镰孢，最低检测灵敏度为 100 pg/μL，是常规PCR检测灵敏度的10倍。表明本研究所建立的LAMP检测体系可快速检测出人工接种和实际发病黄花菜叶片中的病原菌。     

基于Ypt1靶标的大豆疫霉环介导等温扩增技术的研究

大豆疫霉侵染大豆引起的根腐病是大豆生产上的毁灭性病害之一。本研究以Ypt1基因作为靶标,利用环介导等温扩增(LAMP)技术,设计了特异性检测体系,整个过程仅需60 min,即可通过肉眼直接目测检测结果。反应后经浊度仪验证浊度变化、琼脂糖凝胶进行电泳验证和在扩增前加入染料HNB(羟基蔡酚蓝)作为反应指示剂验证扩增结果。特异性检测中,111个大豆疫霉菌株均能产生浊度曲线和扩增到梯形状的条带,同时HNB显色观察到天蓝色的阳性反应,而其它疫霉、腐霉和真菌供试菌株中均没有观察到这些现象;在灵敏度检测中,PsYpt1-LAMP技术最低检测限达到100 pg·μL~(-1),比普通PCR技术的最低检测限高出10倍;在田间应用方面,PsYpt1-LAMP检测技术明显提高了检测效率。本研究建立的LAMP检测体系可用于口岸和田间对大豆疫霉的快速检测。     

李属植物检疫性丁香疫霉和栗黑水疫霉的三重PCR分子检测

为建立我国禁止进境的2种检疫性真菌丁香疫霉Phytophthora syringae和栗黑水疫霉P.cambivora的同步分子检测方法,根据疫霉属的18S rRNA、HSP90和Ypt1基因分别设计通用引物、丁香疫霉和栗黑水疫霉的特异性引物,建立三重PCR检测方法,并进行灵敏度测试和模拟带菌试验。结果表明,可同时检测李属植物上丁香疫霉和栗黑水疫霉的特异三重PCR检测体系为:最佳引物浓度组合18SUF/18SUR、PCSF/PCSR和PSSF/PSSR依次为0.2、0.8、1.0μL,最佳退火温度为63℃,最佳退火时间为20 s。该体系扩增丁香疫霉出现884 bp的18S rRNA条带和683 bp的HSP90基因特异条带,扩增栗黑水疫霉出现884 bp的18S rRNA条带和314 bp的Ypt1基因特异条带,对照菌只出现18S rRNA条带;三重PCR反应体系检测灵敏度低于单重PCR;模拟带菌试验可同时扩增出3个片段。表明该三重PCR检测方法能实现丁香疫霉和栗黑水疫霉的同步特异性检测,可有效改进李属类水果及其种苗上检疫性疫霉的快速检测。     

非洲菊疫霉根腐病的快速分子诊断

 隐地疫霉引起的根腐病是非洲菊生产上的主要病害,为发展该病的快速诊断技术,本文比较了卵菌核糖体基因ITS的序列,在此基础上设计了2条针对隐地疫霉的特异性PCR引物PC1和PC2。供试的23种不同真菌和疫霉菌的46个菌株中,利用这对引物能从隐地疫霉基因组DNA中扩增出一条分子量为620bp的特异性条带,该引物的检测灵敏度可达10pg。采用快速组织碱裂解法提取发病植物组织的DNA,结合PCR检测技术,4h内可从发病的非洲菊根部组织中特异性地检测到隐地疫霉菌。结果表明,建立的非洲菊疫霉根腐病菌分子检测方法可用于该病害的快速分子诊断。     

Tumours of Begonia and some other ornamentals,induced by Corynebacterium fascians

In 1975 many tumours were observed in plants ofBegonia Schwabenland grown in Aalsmeer. Submersion of the roots ofNicotiana megalosiphon seedlings in a homogenate of tumorous tissue, induced tumours after two weeks. Short periods of submergence yielded results similar to those obtained after longer periods. Tumour homogenates lost their infectivity after ten min at 50°C. Aphids transmitted the infectious agent.Treatment with propylene oxide did not inhibit infectivity completely. Filtration through a 450 nm filter removed the infectious agent.Tobacco tumor virus or a viroid could not be isolated. Cultures ofCorynebacterium fascians, isolated from tumours ofN. megalosiphon were highly infectious and induced tumours in healthyN. megalosiphon andBegonia. Tumorous tissue homogenates ofPelargonium zonale, Dahlia sp.,Gladiolus sp., andLilium sp. also caused tumours inN. megalosiphon, from whichC. fascians was isolated. It was not possible to produce tumours inN. megalosiphon with homogenates from roses with symptoms of bud proliferation.Samenvatting In 1975 werden vele tumoren waargenomen inBegonia Schwabenland op Aalsmeerse bedrijven (Fig. 1). De infectiositeit van tumorweefsel kon goed en snel worden vastgesteld door de wortels van zaailingen vanNicotiana megalosiphon in een homogenaat van tumorweefsel te dompelen. Tumoren ontstonden na twee weken, de eindbeoordeling geschiedde na een maand (Fig. 2). Ook verschillende andereNicotiana spp.,Melilotus officinalis (Fig. 3) enPisum odoratum (Fig. 4) werden aangetast.Bij de infectiositeitstoets gaven zeer korte dompeltijden even goede resultaten als langere (Tabel 1). Infectieus sap verloor zijn infectievermogen na 10 min verhitting bij 50°C. Bladluizen brachten de smetstof over. Propyleenoxide verminderde de infectiositeit wel, doch onderdrukte deze niet totaal. Bij filtratie door een 450 nm filter bleef het infectieuse agens op het filter achter. Het tumor-inducerende agens was ook aanwezig in die delen van planten met tumoren welke gezond leken en het ging voor een gering deel over met zaad (Tabel 2).Uit tumoren konden wij geen tabakstumorvirus of een viroïde isoleren. Culturen vanCorynebacterium fascians, geïsoleerd uit tumoren vanN. megalosiphon bleken zeer infectieus en veroorzaakten tumoren inN. megalosiphon enBegonia. Homogenaten van tumorweefsel vanPelargonium zonale, dahlia (Fig. 5), gladiool (Fig. 6) enLilium Mid Century Hybrid Enchantment (Fig. 7) veroorzaakten ook tumoren opN. megalosiphon, waaruitC. fascians werd geïsoleerd. Met sap van kroeskopzieke rozen konden wijN. megalosiphon niet besmetten.     

Epidemiology and simulation of population development of Sitobion avenae in winter wheat

The epidemiology ofSitobion avenae and its natural enemies in winter wheat was studied in 1975, 1976 and 1977. Immigration was important until the end of flowering. The alate immigrants had apterous offspring. These became the driving force in population growth. Their offspring were mostly alatae which usually left the field. A model of the epidemic was developed. Quantitative relations between the aphids and their environment were obtained from literature or established in laboratory trials. The model simulated population development and population composition from the beginning of June till the population peak at the end of June or early in July. Because quantitative data on relations between aphids and their natural enemies and pathogens are scarce, and since the knowledge on wing formation is still limited, the population collapse could not be predicted. In the future, prognosis over a period of three weeks seems possible.Samenvatting De toenemende betekenis van graanbladluizen (vooralSitobion avenae) gepaard gaande met een sterke toename van het gebruik van insecticiden op granen maakte verbetering van de prognose over het schadelijk optreden wenselijk. Door gedetailleerde tellingen in het veld (Fig. 1–7) werden gegevens verkregen over het verloop van de epidemie en het optreden van natuurlijke vijanden in 1975, 1976 en 1977.Een immigratieperiode tot in de bloei kon worden vastgesteld. Daarna lijkt de aantrekkelijkheid van het gewas voor alate luizen te verminderen. De alate immigranten krijgen aptere nakomelingen. Deze vormen de stuwende kracht van de populatiegroei. De nakomelingen van apteren zijn merendeels alaat. Zij verlaten het gewas.Een model van de populatieontwikkeling gedurende de epidemie werd opgesteld. De relatiediagrammen Fig. 9 en 10 laten groei en ontwikkeling vanS. avenae en een predator (Syrphus corollae) zien. Kwantificering van de betrekkingen werd mogelijk door literatuurgegevens en laboratoriumexperimenten.Met het model kon de populatieontwikkeling vanS. avenae vanaf begin juni tot aan de populatiepiek in 1975, 1976 en 1977 vrij goed worden gesimuleerd (Fig. 12). Ook de populatieopbouw kon worden gesimuleerd (Fig. 14). De teruggang van de populatiedichtheid blijkt moeilijker te voorspellen door het ontbreken van gegevens over natuurlijke vijanden.Het lijkt waarschijnlijk dat in de toekomst met het model een prognose over de piek van de bladluispopulatie circa 3 weken tevoren mogelijk zal zijn.     

Inactivation of soil-borne plant pathogens during small-scale composting of crop residues

Samples of heavily infested crop residues were incorporated in static compost heaps (2.5–4.6 m³) of the Indore type. Temperature increased to 50–70°C within 6 days depending on the type of crop residues used and the location within the heap. The heat phase (>40 °C) lasted 2–3 weeks and was followed by a c. 5-months maturation phase (<40 °c).=" among=" the=" 17=" pathogens=" tested,=">Olpidium brassicae and one of the four formae speciales ofFusarium oxysporum that were tested survived composting, but also their inoculum was greatly reduced.Survival during specific phases of composting was studied by incorporation and retrieval of samples at various stages of the process.F. oxysporum f. sp.melonis was completely inactivated andO. brassicae andPlasmodiophora brassicae were almost completely inactivated during the short heat phase. The three pathogens survived the long-lasting maturation phase without loss of viability. Heat evolved during composting was found to be the most important factor involved with sanitation of crop residues. The possible involvement of fungitoxic conversion products and microbial antagonism is discussed.     

A new <Emphasis Type="Italic">Rhizoctonia</Emphasis> sp. closely related to <Emphasis Type="Italic">Waitea circinata</Emphasis> causes a new disease of creeping bentgrass

Isolates of an unidentified Rhizoctonia sp. (UR isolates) were obtained from creeping bentgrass and Kentucky bluegrass with reddish brown sheath and foliar rots. Because the UR isolates anastomosed with isolates of three varieties of Waitea circinata (var. oryzae, var. zeae, and var. circinata), colony morphology, hyphal growth rate at different temperatures, pathogenicity, sequence analysis of the internal transcribed spacers (ITS) region of ribosomal RNA genes (rDNA) were compared. The colony color of mature UR isolates was distinct from isolates of the other three varieties of W. circinata. In pathogenicity tests on creeping bentgrass, the severity of the disease caused by UR isolates was significantly higher than that caused by the three varieties of W. circinata. Sequence similarities of the rDNA-ITS region between UR isolates and between isolates within each variety were high (97–100%), but they were lower among isolates from UR and the varieties of W. circinata (88–94%). In a phylogenetic tree based on the rDNA-ITS sequences, UR isolates formed a cluster separate from each of the clusters formed by the three varieties of W. circinata. These results indicate that the UR isolates clearly differ from the three varieties of W. circinata. We therefore propose that the UR isolates be classified as new Rhizoctonia sp. that are closely related to W. circinata and that the disease on creeping bentgrass should be called Waitea reddish-brown patch disease (Sekikasshoku-hagusare-byo in Japanese).     

Functional analysis of the 3′ end of avrBs3/pthA genes from two Xanthomonas species

The phytopathogens Xanthomonas oryzae pathovar (pv.) oryzae and Xanthomonas axonopodis pv. citri each contain several avrBs3/pthA family genes. Structural features of these genes important for avirulence and/or virulence functions include a central region of multiple direct repeats and three nuclear localization signals (NLSs) and an acidic activation domain (AAD) at the 3′ end. To identify other regions critical to function in the 3′ ends of these genes, we constructed several chimeras using apl1 and apl2 from X. axonopodis pv. citri and avrXa10 and avrXa7 from X. oryzae pv. oryzae and evaluated their functions by inoculation to citrus and rice. The apl1 and avrXa7 genes are major virulence determinants in citrus and rice, respectively, while the contributions of apl2 and avrXa10 to virulence are negligible or not measurable. Constructs that contained a 417 bp HincII-SphI fragment from the 3′ end of apl1 in combination with the repeats from avrXa7, avrXa10, and apl1 caused a canker phenotype on citrus. Interchange of the HincII-SphI fragment between avrXa7 and avrXa10 abolishes avrXa7 avirulence function and reduces its virulence but it does not affect avrXa10 avirulence function in rice. avrXa7 caused a hypersensitive response (HR) in citrus and replacement of it's 3′ end with that of apl1 resulted in loss of canker and induction of HR. Thus, the HincII-SphI fragment of the avrBs3/pthA gene family is important for avirulence and virulence functions in two different plant species, Oryza sativa and Citrus natsudaidai HAYATA.     

Identification,incidence and characterization of <Emphasis Type="Italic">Fusarium proliferatum</Emphasis>on ornamental palms in Spain

During a survey conducted from 1998 to 2002, Fusarium proliferatum was found associated with young and adult palms belonging to the genera Chamaerops, Phoenix, Trachycarpus and Washingtonia showing symptoms of wilt and dieback. The fertility and toxicological profile of 36 strains representing different locations and hosts were studied. All of them except two, which were infertile, belonged to mating population D. Both mating types (MATD-1 and MATD-2) were isolated from the same host species, showing a high potential of genetic recombination in the field. Additionally, eight strains were fertile once crossed as female. Toxin analysis showed differences in the ability of strains to produce fumonisin B₁, moniliformin, beauvericin, fusaric acid and fusaproliferin. Only 17 of them produced all the toxins analyzed. Pathogenicity tests were conducted on Phoenix dactylifera and P. canariensis using nine F. proliferatum Spanish strains and two reference strains from Saudi Arabia. Eight months after inoculation all strains caused disease, with palms showing lesions on the bases of leaves and development of wilt symptoms similar to those originally observed in affected plants. This is the first report on the occurrence of F. proliferatum on P. dactylifera in Spain and also the first report of this pathogen on C. humilis, P. canariensis, P. reclinata, T. fortunei, W. filifera and W. robusta.     

Nematicidal Activity of Chrysanthemum coronarium

Organic amendments and green manure are potential alternatives to the harmful chemical control means currently used against plant-parasitic nematodes. In this work, Chrysanthemum coronarium was applied to the soil as a green manure to control the root-knot nematodes Meloidogyne incognita and M. javanica. Chrysanthemum coronarium significantly reduced nematode infection of tomato roots and improved plant-top fresh weight, both in the greenhouse and in microplots. Other green manures, derived from Anthemis pseudocotula, wild chickpea (Cicer pinnatifidum), Geranium spp. and wheat, were not as effective as C. coronarium. Chrysanthemum coronarium, retained its nematicidal activity even when applied as a dried material. Only mature C. coronarium plants, in their flowering stage, exhibited nematode control activity, but the green plant parts were more effective than the flowers. An aqueous extract of C. coronarium exhibited in vitro, nematostatic activity towards M. incognita and M. javanica second-stage juveniles and inhibited their hatching from eggs and egg-masses; its nematostatic activity was expressed also against other phytonematode species such as Heterodera avenae and Pratylenchus mediterraneus, but did not affect the beneficial entomopathogenic nematode Steinernema feltiae.     

Pathogenic fungi involved in root rot of peas in the Netherlands and their physiological specialization

Research on root rot pathogens of peas in the Netherlands has confirmed the prevalence ofFusarium solani, F. oxysporum, Pythium spp.,Mycosphaerella pinodes andPhoma medicaginis var.pinodella. Aphanomyces euteiches andThielaviopsis basicola were identified for the first time as pea pathogens in the Netherlands. Other pathogens such asRhizoctonia solani andCylindrocarpon destructans were also found on diseased parts of roots. F. solani existed in different degrees of pathogenicity, and was sometimes highly specific to pea, dwarf bean of field bean, depending on the cropping history of the field.A. euteiches was specific to peas, whereasT. basicola showed some degree of physiological specialization.     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454     

Identification and pathogenicity of Pythium species causing damping-off of kidney bean

Five Pythium species (Pythium irregulare, P. mamillatum, P. myriotylum, P. spinosum and P. ultimum var. ultimum) were isolated from the hypocotyls and roots of kidney bean plants with damping-off from a commercial field and from experimental plots that have undergone either continuous cropping with kidney bean or rotational cropping with arable crops. In inoculation tests, all five Pythium species were pathogenic to kidney bean. This is the first report of damping-off of kidney bean caused by Pythium species; we named this disease damping-off of kidney bean. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB291811, AB291944 and AB291945.