Dry-dispersal and rain-splash of brown (Puccinia recondita f.sp. tritici) and yellow (P. striiformis) rust spores from infected wheat leaves exposed to simulated raindrops

The dispersal of spores from lesions of brown ( Puccinia recondita f.sp. tritici ) or yellow ( P. striiformis ) rusts of wheat by impacting drops was studied. Using a generator of uniform-size drops, drops of 2.5, 3.4, 4.2 and 4.9 mm in diameter were released from rest at heights of 5, 50 and 100 cm above horizontal and primary leaves uniformly covered with sporulating lesions. Dry-dispersal and rain-splash occurred simultaneously in response to drop impaction. A coloration technique allowed separate counting of dry-dispersed and rain-splashed spores caught on slides. More spores were rain-splashed than dry-dispersed. Neither removal mechanism affected in-vitro germination of spores, which was higher in brown than in yellow rust. For both rusts, the number of both dry-dispersed and rain-splashed spores, as well as their travel distance, increased with drop diameter and fall height. The fall speed of incident drops in relation to diameter and fall height was obtained by solving numerically the equation of vertical drop motion. The number of spores removed by a given impacting drop was found to be a power function of the calculated kinetic energy of the impacting drop. Based on this experimental relationship, a simulation study showed the relevance of rain type in the removal of spores.     

Local dispersal of Puccinia striiformis f. sp. tritici from isolated source lesions

Understanding how disease foci arise from single source lesions has not been well studied. Here, single wheat leaves were inoculated with Puccinia striiformis f. sp. tritici urediniospores, and all wheat leaves within two intersecting 0.3 × 3.0 m transects were sampled in eight replicates over 3 years. The lesions observed on each of the top three leaves on plants within 1.5 m from the source lesion were three‐dimensionally mapped. The total number of lesions within a 1.5 m radius was estimated by dividing the number of lesions observed within each 0.025 m‐wide annulus by the fraction of the annulus sampled. The estimated total number of lesions produced within 1.5 m of a single source lesion ranged from 27 to 776, with a mean of 288 lesions. Eighty percent of the lesions were recorded within 0.69 m of the source infection. The proportion of total lesions observed at a given distance from the source was fitted well by the Lomax and Weibull distributions, reflecting the large proportion of lesions arising close to the source, and when fitted by an inverse power distribution had a slope (b) of 2.5. There were more lesions produced on leaves higher in the canopy than on lower leaves, with more lesions being detected above than below the point of inoculation. Simultaneous measurement of lesion gradients and spore dispersal in the final year of the study suggests that this pattern is due to greater susceptibility of upper leaves, rather than increased dispersal to upper leaves.     

Clonality and long-distance migration of Puccinia striiformis f.sp. tritici in north-west Europe

The biotrophic fungus Puccinia striiformis f.sp. tritici , a basidiomycete that causes yellow rust on wheat, is spread by wind-dispersed spores. Analysis of amplified fragment length polymorphism (AFLP) variation showed that the fungus frequently migrates between the UK, Germany, France and Denmark. There is no biological evidence for sexual or parasexual reproduction under natural conditions, and this was supported by the lack of recombination, as revealed by AFLP, over the time and area represented by the samples in this study. A phylogeographic analysis revealed that there was effectively a single, clonal population in the four countries, up to 1700 km apart, consistent with a 'continent-island' model in which Denmark is the recipient of migrants from other countries. In five cases, specific pathogen clones were dispersed between the UK and Denmark, and on at least two recent occasions clones were also spread from the UK to Germany and France, causing outbreaks of yellow rust on wheat cultivars that were previously resistant to the disease in these countries. The agronomic consequences of migration were enhanced because of the limited genetic diversity for yellow rust resistance in wheat cultivars in the area. These results demonstrate that long-distance migration of pathogen clones, coupled with low diversity in the host species, may cause previously useful resistance genes to become ineffective for disease control on a continental scale.     

小麦叶锈菌在感病寄主上发育的组织病理学和超微结构研究

 应用荧光显微技术、微分干涉技术和生物电镜技术,系统地研究了小麦叶锈菌在感病寄主上的发育过程及其超微结构特征。小麦叶锈菌在感病品种上的发育过程可分为几个明显的阶段,即孢子的萌发、附着胞的形成、气孔下囊的分化、初生菌丝和次生菌丝的形成和生长、吸器母细胞和吸器的形成、夏孢子床和夏孢子堆的产生以及夏孢子的形成。小麦叶锈菌的胞间菌丝呈丝状,生长和分枝通常沿寄主细胞壁进行。胞间菌丝与寄主细胞的接触诱导了吸器母细胞的分化,吸器母细胞在与寄主细胞壁的接触部位发育形成入侵栓,穿透寄主细胞壁后于细胞内形成吸器。胞间菌丝和吸器母细胞均含有双核,而成熟吸器则含有单核。经常规染色后,胞间菌丝和吸器母细胞的壁与隔膜均可分辨出由多层构成。     

我国小麦秋苗条锈病发生规律及其区间菌源传播关系

全国冬小麦秋苗均可遭受来自西北和西南越夏区条锈菌的侵染与危害, 条锈菌在寄主上进一步繁殖和发展, 引起本地或者外地小麦条锈病流行。不同麦区秋苗条锈病发生时间、发生程度及其菌源传播规律各不相同, 年度间亦存在差异。根据小麦条锈病发生流行频率、病菌越冬和越夏情况、秋季菌源和春季菌源的有无与多少、提供时间及其影响范围与作用, 结合地理生态条件、气候特点、小麦种植区划与栽培模式等, 将中国小麦条锈病发生流行区域划分为8个明显不同的生态区系, 即关中、华北春季流行区; 成都平原、江汉流域冬季繁殖区; 西北、川西北越夏易变区; 云贵高原越夏冬繁区; 新疆冬春麦常发区; 西藏高原青稞、小麦常发区; 南方晚播冬麦偶发区; 内蒙古、东北春麦偶发区。通过病害实地调查、病菌群体遗传多样性和高空气流轨迹分析, 进一步揭示了区间菌源传播关系。陇南、陇中、陇东、宁南、海东、陕西宝鸡以及川西北和云贵高原等地区离越夏区较近, 冬小麦播种较早, 秋苗条锈病发生早、发病重, 秋季随西北气流传播到平原冬麦区和海拔较低的冬麦区侵染危害秋播麦苗, 其菌源数量对全国小麦条锈病发生流行程度起着至关重要的作用, 是中国小麦条锈病的秋季菌源基地, 面积约67万hm2; 成都平原、江汉流域、陕南、豫南、云贵坝区等麦区, 离条锈菌越夏区相对较远, 小麦播种期也较晚, 秋苗发病较晚较轻, 但冬季气候温和, 雨露条件充沛, 条锈菌在冬季可以不断侵染和繁殖, 在早春可积累大量菌源, 然后向北部和西部广大麦区扩散传播, 引起小麦条锈菌春季侵染, 是中国小麦条锈病的春季菌源基地, 面积约200万hm2。云贵高原越夏冬繁区可为我国广大麦区特别是长江中下游麦区提供部分菌源, 荆州等鄂东南地区是云贵菌源向长江下游麦区传播的中转站。云南与甘肃之间存在大量的基因流, 推测云南可能是中国小麦条锈菌重要的起源中心。西南和西北秋季菌源对长江流域麦区的相对重要性有待进一步研究明确。     

Histological investigation of stripe rust (Puccinia striiformis f.sp. tritici) development in resistant and susceptible wheat cultivars

The wheat cultivar Kariega expresses complete adult plant resistance against stripe rust, whereas cv. Avocet S is susceptible. Using confocal laser scanning microscopy, initial fungal penetration into flag leaves was identical in both cultivars, with directional germ-tube growth towards stomata that were penetrated without the formation of an appressorium, followed by differentiation of a substomatal vesicle, infection hyphae, haustorial mother cells and haustoria. During the following 4 days, further fungal development occurred more quickly in the resistant than in the susceptible cultivar. However, by 7 days postinoculation (dpi) the situation changed, with exponential growth of the pathogen occurring only in the susceptible line. Induced cellular lignification, a typical defence reaction of cereals, was observed at 4 dpi in the resistant cultivar, and 2 days later lignified tissue completely surrounded the fungal colonies. In the susceptible cultivar, isolated lignified host cells occurred at 6 dpi, and long, unbranched fungal hyphae outgrowing the resistance reaction were observed.     

Effects of growth stage, leaf position and temperature on adult-plant resistance of wheat infected by Puccinia recondita f.sp. tritici

Growth stage, leaf position and temperature effects on components of resistance to Puccinia recondita f.sp. tritici in wheat ( Triticum aestivum ) were studied. Inoculation of Era, Glenlea, Sinton and RL6044 plants at five growth stages showed that latent period increased and uredinium numbers decreased as each adult-plant-resistant wheat matured. Growth stage had little effect on latent period in the susceptible cultivar Morocco. No significant differences in latent period determined on the flag, flag -1 and flag -2 leaves of the same plant were observed for Era, Glenlea and RL6044. However, leaf age should be considered in assessing adult-plant resistance because the latent period of P. recondita f.sp. tritici in flag leaves of Sinton was shorter than that determined for the flag-1 and flag -2 leaves. Fewer uredinia developed on the flag -2 leaf of Glenlea than on the flag leaf. The latent period of leaf rust in Era, Glenlea, RL6044 and Morocco was significantly longer at 15°C than at 21°C, but temperature did not affect Sinton to the same degree. Uredinium size was a sensitive criterion of adult-plant resistance. Uredinium development in line RL6044 was significantly restricted at 15°C, but uredinia on Era, Glenlea and Sinton at 15°C were as large as those at 21°C. On the susceptible Morocco uredinia were larger at 15°C than at 21°C.     

Effect of interrupted leaf wetness periods on pustule development of Puccinia recondita f. sp. tritici on wheat

In growth chamber experiments, seedlings of a susceptible wheat cultivar (Rubis) were inoculated with urediospores of wheat leaf rust. Inoculated seedlings were incubated in a moist chamber. The period of exposure to leaf wetness varied in duration and progressed with or without one or more interruptions of variable duration. During the interruptions the leaves with germlings were dry. At near-optimal temperature, leaf wetness periods of six h resulted in 60 to 65% of the pustules produced with 12 or 24 h wetness periods. Interruption of a 6 h leaf wetness period by a 1 h dry period was most damaging to the rust germlings at about three to four h after inoculation. The damage became visible as a reduction in the number of resulting pustules and as a prolongation of the median latency period. Epidemiological implications of leaf wetness interruptions are discussed.Kiemplanten van het vatbare tarweras Rubis werden geïnoculeerd met uredosporen van de bruine roest van tarwe. De proeven vonden plaats in klimaatkamers. Geïnoculeerde planten werden geïncubeerd in incubatiehokjes met een zeer hoge luchtvochtigheid, teneinde condensatiedruppels op de bladeren en aldus bladnatperioden te krijgen. De toegediende bladnatperioden varieerden in tijdsduur en zij verliepen met of zonder één of meer onderbrekingen, ook weer van variërende duur. Tijdens de onderbrekingen stonden de planten met roestkiemlingen in het licht; het blad was dan droog. Bij ongeveer optimale temperatuur en bladnatperioden van 6 uur was het aantal verkregen puistjes 60 à 65% van het aantal verkregen puistjes na een bladnatperiode van 12 of 24 uur. Onderbreking van de 6-urige bladnatperiode door een 1-urige droge periode was voor de roestkiemlingen het schadelijkst ongeveer 3 tot 4 uur na inoculatie. De schade kwam tot uitdrukking in het kleinere aantal geproduceerde puistjes en in een verlenging van de mediane latentieperiode. De epidemiologische betekenis van onderbrekingen van de bladnatperiode wordt besproken.     

小麦条锈菌cDNA文库构建和表达序列标签(ESTs)分析

 小麦条锈菌(Puccinia striiformis f. sp. tritici)是全世界范围内小麦生产上的重要病原真菌, 但是对小麦条锈菌的基因组和基因功能却了解甚少。为了促进小麦条锈菌基因组学的发展和大规模基因发现, 我们以噬菌体λTrip1Ex2为载体, 采用SMART技术构建了小麦条锈菌萌发夏孢子的cDNA文库。原始文库的滴度为1.1×10⁶pfu/mL, 平均插入片段长度为750 bp。从文库中随机挑取279个cDNA克隆测序, 分析发现这些ESTs的平均GC含量为45.08%。通过聚类分析, 279个ESTs拼接成31个contigs和80 singletons。BLASTx分析表明, 47%的ESTs与GenBank中报道的功能已知或未知蛋白具有相似性。tBLASTx分析表明12个uniseqs与EST数据库中的序列具有相似性, 其中9个是来自担子菌的cDNA文库。几个EST与已知的真菌致病相关基因具有高度相似性。RT-PCR分析了几个基因在小麦条锈菌侵染过程中的表达水平。这些结果为小麦条锈菌夏孢子萌发以及侵染寄主过程中的基因表达研究奠定了很好的分子生物学基础。     

普通小麦品种陕旱8675中抗条锈病基因的分子定位

 利用SSR标记技术对小麦品种陕旱8675的抗条锈病基因进行了分子标记定位。通过对290对微卫星引物的筛选,发现Xwmc170和Xcfa20432对引物在抗亲、感亲、抗池和感池之间均有多态。群体分析结果表明,Xwmc170和Xcfa2043与陕旱8675中抗病基因相连锁,遗传距离分别为9.8 cM和15.0 cM,基因和标记之间的顺序为着丝点-Xcfa2043-Xwmc170-YrSh,间隔距离分别为19.05、5.2和9.8 cM。根据作图的结果,将陕旱8675中所含有的抗病基因定位于2A染色体长臂上,根据该基因的作图位置与抗谱分析,认为该基因可能是1个新的抗条锈基因,暂定名为YrSh。     

条锈菌诱导的小麦EF手钙离子绑定蛋白基因TaCab1的功能初步分析

 根据小麦EF手钙离子绑定蛋白(TaCab1)基因序列,利用WMD3软件设计特异的人工miRNA (amiRNA),构建VIGS沉默载体。利用amiRNA-VIGS体系,对小麦的TaCab1基因的功能进行了初步分析。利用Northern blot和实时定量PCR技术分别检测了amiRNA的积累及TaCab1的沉默效率,并利用显微观察技术统计条锈菌侵染小麦后的组织学差异。结果表明,amiRNA可以得到有效的积累,其靶标基因TaCab1可以得到有效的沉默。从表型上看,小麦叶片上条锈菌夏孢子的产孢量也在一定程度上有所降低。组织学观察发现当TaCab1被沉默后,寄主细胞的坏死面积在侵染后期明显增大,条锈菌的菌丝分枝数也明显增多,但菌丝长度明显变短。     

Relationships and genetics of wheat effects on infection frequency and colony extension of Puccinia striiformis f. sp. tritici

Wheat cvs Aquileja and Xian Nong 4, previously reported to possess quantitative resistance to Puccinia striiformis f. sp. tritici (Pst), were crossed to assess resistance associated with infection frequency and colony extension of Pst. The parents, F ₁, F ₂, F ₃, F ₄, backcrossed to Aquileja, and backcrossed to Xian Nong 4 were sown in the field. Experimental plants were inoculated with a single-spore derived isolate of Pst. Penetration frequency, colony size and disease severity were quantified. Comparison between the parents indicated that Aquileja was better at limiting infection frequency of Pst, whereas Xian Nong 4 was better at restricting colony extension. Penetration frequency and colony size were controlled by different genes. It was estimated that three or four genes, and three genes, controlling penetration frequency and colony size respectively, segregated in the cross Aquileja × Xian Nong 4, and were transmitted with moderately high heritability in additive fashion with no detectable dominant and epistatic effects. The two traits were correlated with disease severity. Colony size and penetration frequency explained, respectively, up to 33 and 8% of the genotypic variation of disease severity. These results will be helpful in understanding quantitative resistance and breeding for enhanced resistance to Pst.     

利用小种标记分析云南省两个县的小麦条锈菌群体

 小麦条锈病是由小麦条锈菌（Puccinia striiformis f.sp. tritici）引起的气流传播病害,在世界各主要麦区均有发生,也是我国小麦生产上危害最严重的病害之一。小麦条锈菌毒性变异频繁,易产生新毒性小种,导致小麦品种的抗病基因失效,引起小麦条锈病周期性流行。因此,了解小麦种植区的条锈菌群体结构组成及变异,对于制定更为有效的条锈病控制方法具有重要的意义。     

Nitrogen per unit leaf area affects the upper asymptote of Puccinia striiformis f.sp. tritici epidemics in winter wheat

Field trials tested which components of epidemic development of Puccinia striiformis , the cause of yellow rust, were affected by nitrogen (N) fertilizer applied to winter wheat. Both timing and amount of N were varied to affect canopy size and leaf N content, and to provide a supply of mobile N to the pathogen, by causing fresh N uptake after leaf expansion was complete. No N was applied to control plots. A logistic disease-progress function was fitted to disease-severity data, which were assessed in absolute units. Leaf area and specific leaf N (g N per m² leaf tissue) were quantified. Large and highly significant effects of N on the upper asymptotes, or 'carrying capacities' ( c ) were found. Effects on rates and points of inflection of the epidemics were not significant. Early N resulted in larger shoot numbers and leaf area, but disease was also more severe, so that by grain filling, the remaining green leaf areas were larger without N than with N. Later N treatments did not increase canopy size, but did increase symptom area compared with the control. These effects differ from the concept that N affects disease as a result of its effect on canopy growth, and therefore canopy microclimate, and suggest instead a substrate effect. Linear regression revealed that 51% of the observed differences in c were explained by variation in specific leaf N, suggesting that growth of the rust fungus may depend directly on particular components of total leaf N.     

小麦-条锈菌互作过程中活性氧及保护酶系的变化研究

 对条锈菌与小麦不同互作体系中组织学特征、活性氧产生及其相关酶系的变化进行了研究。组织学观察表明:非亲和组合相对于亲和组合存在明显差异,表现为菌丝生长受抑,吸器母细胞和吸器形成减少,寄主细胞在接种后18h左右出现过敏性坏死。生化测定结果表明:在非亲和组合中,O₂^-的产生速率和H₂O₂的含量均高于亲和组合,且O₂^-产生速率在接种后12h达到一个峰值,H₂O₂的含量在接种后20和72h出现2个高峰。而在亲和组合中,O₂^-产生速率低于对照或与对照相似,H₂O₂的含量虽然高于对照,但却普遍低于非亲和组合;SOD在亲和组合中的活性总体上要高于非亲和组合;接种24h后,CAT在2种组合中的活性均高于对照,在接种后36和48h时,亲和组合中的CAT活性高于非亲和组合,而在接种60h后又开始低于非亲和组合;POD活性在接种24h后均明显升高,但亲和组合中POD活性增幅大;MDA在非亲和组合中于接种后72h含量明显上升。结果表明,亲和与非亲和组合中条锈菌扩展、活性氧的产生及相关酶活变化都存在明显差异,这些差异与小麦抗锈性的表达可能有密切联系。     

小麦条锈菌hsp70基因的克隆及热胁迫下的表达特征分析

为明确热激蛋白70在小麦条锈菌对高温适应性中的作用,采用RACE和PCR技术扩增得到小麦条锈菌hsp70的基因全长,并利用实时荧光定量PCR技术对热胁迫下不同温度敏感类型小麦条锈菌中hsp70的表达特征进行分析。克隆得到的小麦条锈菌hsp70基因组序列全长为2 817bp,包含7个内含子,cDNA序列全长为2 267bp,其中开放阅读框为1 917bp,编码639个氨基酸,分子量为66.7ku,等电点为5.15。编码蛋白含3个保守的HSP70氨基酸序列。qRT-PCR试验结果表明,28℃热激后,hsp70的转录水平随热激时间的延长而略有增加,2h达到最高值。不同温度敏感类型菌株在热激2h后hsp70的相对表达量具有显著差异。低敏感类型菌株hsp70平均表达量为未经处理对照的7.12倍,而高敏感类型菌株hsp70平均表达量仅为对照的1.63倍。     

小麦条锈病跨区域全周期绿色防控技术体系的构建与应用

小麦条锈病是我国小麦生产上一种跨区域流行的重大病害。1949年以来, 我国小麦条锈病研究, 治理能力明显提升, 发病面积、流行程度、流行频率明显降低, 但由于条锈菌Puccinia striiformis f.sp. tritici新毒性小种不断产生和气候异常变化等因素的影响, 近年来其流行规律和成灾机制出现了新的变化, 发生危害加重, 严重威胁小麦生产和国家粮食安全。基于“十二五”以来我国在小麦条锈病菌毒性新小种产生、有性生殖、侵染循环和大区流行规律等方面的最新研究成果, 作者在学习和实践以往病害治理经验的基础上, 坚持长短结合、标本兼治、分区治理、综合防治的指导思想, 制定了“以绿色防控为基础, 以全周期管理为重点, 以跨区域联防联控为保障”的防控策略, 提出了精准监测预报、毒性小种变异监测、早期菌源控制、抗病品种合理布局和应急防控等关键技术, 集成构建了全国小麦条锈病跨区域全周期绿色防控技术体系, 制定了越夏易变区、冬季繁殖区(越冬区)和春季流行区分区防控技术体系。经在小麦条锈病主要流行区大面积试验示范, 取得了防控效果85%以上, 化学农药用量减少10%以上, 小麦单产提高5%以上的成效。2022年-2023年在全国小麦主产区大范围组织推广应用, 有效控制了小麦条锈病严重流行势头, 全国病害发生面积出现了新低, 有效减轻了灾害损失, 保障了国家粮食安全。     

Predicting epidemics of yellow rust (Puccinia striiformis) on the upper canopy of wheat from disease observations on lower leaves

Data from field experiments were used to test whether disease observations on lower leaves of wheat were good predictors of future epidemic development in the upper canopy. Fungicide treatments to replicated plots in 1994 and 1995 caused variation in levels of initial inoculum of urediniospores of yellow rust ( Puccinia striiformis ). Observations of symptom severity were made on individual leaf layers throughout the season. Sporulating lesions on lower leaves were considered to be a measure of inoculum source strength for transfer to upper culm leaves. Low source strengths were associated with delays in epidemic development on the upper leaves, as quantified by a location parameter, t _m, the time at which severity reached half of the asymptote value in logistic fits to disease progress data on the upper leaves. However, there was much unexplained variation, probably due to extraneous variation in upward transfer efficiency of inoculum and rates of epidemic development. Vanderplank's sanitation ratio theory was used to account for variation in inoculum transfer and epidemic rate. The analysis revealed that if the aim is to predict disease on a newly emerged culm leaf, during the period when it can be treated effectively with fungicide, then observations of disease two leaves further down the culm were of the greatest predictive value ( R ² = 86%). These observations, however, need to be integrated with information about factors affecting upward inoculum transfer and rates of epidemic development if acceptable predictive precision is to be achieved.     

Pathogenomic analyses of Puccinia striiformis f. sp. tritici supports a close genetic relationship between South and East Africa

Wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), occurs in all major wheat-growing regions worldwide and poses a constant threat to production. In South Africa, Pst first emerged in 1996 in the Western Cape and has since caused frequent epidemics with three further distinct races (pathotypes) recorded to date. Herein, we undertook detailed genomic-based analyses of four Pst isolates that represent the four dominant Pst races in South Africa recorded between 1996 and 2005. This analysis identified a number of polymorphic genes with features of known effector proteins and provided additional support of the likely stepwise changes in virulence profile of these South African Pst isolates. Next, we carried out comparative genomic-based analyses with 54 additional Pst isolates collected across wheat-growing regions within South Africa between 1996 and 2017 and 58 Pst isolates from East Africa, Pakistan, the UK, and France. This revealed a close genetic relationship between Pst isolates in South Africa and a number from East Africa. Furthermore, we found the South African Pst isolates also grouped closely with isolates identified in the UK in 2013 that were specifically found on triticale, illustrating long-distance transmission of Pst isolates either between these regions or from a common independent source area. This highlights the critical need for close monitoring of Pst. With wheat being the most planted winter cereal crop in South Africa, investment in continuous surveillance is essential to rapidly identify any future introductions that could quickly lead to rust epidemics.     

Support for a stepwise mutation model for pathogen evolution in Australasian Puccinia striiformis f.sp. tritici by use of molecular markers

Since its initial detection in Australia in 1979, wheat yellow (stripe) rust ( Puccinia striiformis f.sp. tritici ) has evolved in Australia and New Zealand into more than 20 pathotypes with assorted virulence characteristics. This evolution is believed to have occurred in a stepwise fashion from an original single pathotype, with no subsequent new introductions. A combination of random amplified polymorphic DNA (RAPDs) and amplified fragment length polymorphisms (AFLPs) was used to examine the level of molecular variation in Australian and New Zealand isolates, and to compare this with variation amongst other isolates of P. striiformis . Using 60 RAPD primers on seven Australian isolates representing seven different pathotypes collected between 1979 and 1991, more than 300 potentially polymorphic loci were analysed and no polymorphisms were detected. Using the same primers on two UK isolates, 3% of loci showed a polymorphism. A similar level of polymorphism was found between UK isolates using AFLP primers, and between 5 and 15% of fragments were polymorphic between an isolate from the UK, an isolate from Denmark, and one from Colombia. However, no AFLP polymorphisms were found amongst 14 Australian and New Zealand isolates tested, at over 100 potentially polymorphic loci. The lack of molecular variation in the Australian and New Zealand collection is consistent with the stepwise mutation theory of pathotype evolution from a single introduction.