A comparative study of Turkish and Israeli populations of Didymella rabiei,the ascochyta blight pathogen of chickpea

The Fertile Crescent is the centre of domestication of chickpea (Cicer arietinum) and also the place of origin of its pathogens. Agrosystems provide different environments to natural eco‐systems, thus imposing different types of selection on pathogens. Here, the genetic structure and in vitro temperature growth response of the chickpea pathogen Didymella rabiei from domesticated chickpea (59 isolates from Turkey and 31 from Israel) and wild Cicer spp. (three isolates from Turkish C. pinnatifidum and 35 from Israeli C. judaicum) were studied. Six sequence‐tagged microsatellite site (STMS) primer pairs were used to determine the genetic structure of the 128 D. rabiei isolates. Turkish isolates exhibited the highest genetic diversity (H = 0·69). Turkish and Israeli D. rabiei from domesticated chickpea were genetically closer to each other than isolates from the wild Cicer spp. Analysis of molecular variance showed that 54% of the genetic variation resided between isolates from wild and domesticated origins. EF1‐α sequences distinguished between D. rabiei isolates from domesticated and wild Cicer spp. by four polymorphic sites. Nevertheless, a certain degree of mixing between isolates from wild and domesticated origin was demonstrated using the Bayesian algorithm as well as with principal coordinates analysis. Isolates sampled from domesticated chickpea from both countries were better adapted to temperatures typical of Levantine spring and had a significantly larger colony area at 25°C than at 15°C (typical Levantine winter temperature). These observations were in accordance to the heritability values of the temperature growth response.     

Ascochyta blight of chickpea in Australia: identification, pathogenicity and mating type

The aetiology of blight of chickpea in South Australia was studied following sporadic disease outbreaks over several years that had been tentatively identified as Phoma blight. Nine fungal isolates from diseased chickpeas were tested for pathogenicity in the glasshouse, of which two caused symptoms resembling those of Ascochyta blight. The two aggressive isolates were identified as Ascochyta rabiei based on morphological characteristics of cultures and RAPD analysis. This was further confirmed by successful mating to international standard isolates, which showed that the two Australian isolates were MAT1-1. These isolates are accessioned as DAR 71767 and DAR 71768, New South Wales Agriculture, Australia. This is the first time that A. rabiei has been positively identified in commercial chickpeas in the southern hemisphere. The pathogen was found (in 1992) in only one of 59 seed samples harvested throughout Australia between 1992 and 1996 and tested using International Seed Testing Association methods. The teleomorph has not been found in Australia and results to date suggest that only one mating type is present. This suggests that quarantine restrictions on imported chickpea seed should be retained to prevent the introduction of the opposite mating type.     

新疆鹰嘴豆壳二孢疫病病菌Ascochyta rabiei交配型、致病力及其对3种杀菌剂的敏感性测定

新疆是我国鹰嘴豆主要生产地,然而随着鹰嘴豆种植面积的扩大,集约化种植模式的发展以及广泛从国内外引种,鹰嘴豆壳二孢疫病(Ascochyta blight)开始发生。由于该病害在新疆发生历史较短,相关的研究还甚少。本研究对新疆鹰嘴豆壳二孢疫病病菌Ascochyta rabiei的交配型、致病力及对3种杀菌剂敏感性进行测定,结果表明,A.rabiei的交配型MAT1-1和MAT1-2在新疆木垒地区的分离频率明显偏离1∶1,以MAT1-2为优势种群,但其致病力与MAT1-1无显著差异,而MAT1-1对30%醚菌酯EC的敏感性显著低于MAT1-2。     

Population structure and mating system of Ascochyta rabiei in Tunisia: evidence for the recent introduction of mating type 2

The population structure of Ascochyta rabiei (teleomorph: Didymella rabiei ) in Tunisia was estimated among five populations sampled from the main chickpea growing regions using simple sequence repeat markers (SSR) and a mating type ( MAT ) marker. Mating type 2 isolates ( MAT1-2 ) had reduced genetic and genotypic diversity relative to mating type 1 isolates ( MAT1-1 ). This result, coupled with previous observations of lower overall frequency and restricted geographical distribution of MAT1-2 in Tunisia, and recent (2001) observation of the sexual stage, support the hypothesis of a recent introduction of MAT1-2 . Despite the presence of both mating types in Nabeul, Kef and Jendouba, the hypothesis of random mating was rejected in these locations with multilocus gametic disequilibrium tests. Highly significant genetic differentiation ( θ  = 0·32, G _ST = 0·28, P  < 0·001) was detected among populations and genetic distance and cluster analyses based on pooled allele frequencies revealed that populations from Nabeul and Kef were distinct from those in Beja, Bizerte and Jendouba. More than 70% of total gene diversity ( H _T = 0·55) detected was attributable to variation within populations compared to 28% among populations. This result, coupled with the occurrence of private alleles in each population, suggests that gene flow is currently limited among populations, even those separated by short geographic distances. The presence of two main genetic clusters was confirmed using Bayesian model-based population structure analyses of multilocus genotypes (MLGs) without regard to geographic origin of samples. The presence of MAT1-2 isolates in both clusters suggests at least two independent introductions of MAT1-2 into Tunisia that are likely to be the result of importation and planting of infected chickpea seeds.     

Mating type, pathotype and RAPDs analysis inDidymella rabiei, the agent of ascochyta blight of chickpea

Eleven pathotype groups (A-K), including five not previously reported, ofDidymella rabiei (anamorphAscochyta rabiei), representing isolates of the pathogen from Ascochyta blight-affected chickpeas mainly from India, Pakistan, Spain and the USA, were characterized using 44 single-spore isolates tested against seven differential chickpea lines. Of 48 isolates tested for mating type, 58% belonged to MAT 1-1 and 42% to MAT 1-2. Thirty-nineD. rabiei isolates, as well as two isolates ofAscochyta pisi and six isolates of unrelated fungi, were analyzed using Randomly Amplified Polymorphic DNAs (RAPDs) employing five primers (P2 at 40°C, and OPA3, OPC1, OPC11 and OPC20 at 35°C). Computer cluster analysis (UPGMA / NTSYS-PC) detected a relatively low level of polymorphism among all theD. rabiei isolates, although atca 7% dissimilarity,ca 10 RAPD groups [I-X] were demarcated, as well as subclustering within the larger groups. By the same criteria, the maximum dissimilarity for the whole population ofD. rabiei isolates wasca 13%. No correlation was found between different RAPD groups, pathotype, or mating type ofD. rabiei, although some evidence of clustering based on geographic origin was detected. The use of RAPDs enabled us to identify specific DNA fragments that may have a potential use as genetic markers in sexual crosses, but none which could be used as virulence markers.     

Development of ascochyta blight (Ascochyta rabiei) in chickpea as affected by host resistance and plant age

Ascochyta blight caused by Ascochyta rabiei, is the most destructive disease in many chickpea growing countries. Disease development varies with the growth stage and host resistance. Hence, disease development was studied in cvs ICCX 810800 (resistant), ICCV 90201 (moderately resistant), C 235 (moderately susceptible), ICCV 96029 and Pb 7 (susceptible) under controlled environment (ICRISAT, Patencheru) and field conditions (Dhaulakuan, Himachal Pradesh) at seedling, post-seedling, vegetative, flowering and podding stages. Under controlled environment, the incubation period and terminal disease reaction (TDR) did not vary significantly at different growth stages against virulent isolate AB 4. Cultivars ICCX 810800, ICCV 90201 and C 235 showed a significantly longer incubation period than the susceptible cv. Pb 7. Cultivar ICCX 810800 showed slow disease progress and the least TDR. Field experiments were conducted during the 2003–2004 and 2004–2005 growing seasons. During 2003–2004, TDR was higher in plants inoculated at podding and the flowering stage and the lowest disease reaction was recorded in ICCX 810800. A severe epidemic during 2004–2005 was attributed to the favourable temperature, humidity and well distributed high rainfall. TDR did not differ significantly at any of the growth stages in susceptible cvs ICCV 96029 and Pb 7. With respect to seeding date and cultivar, the highest yield was recorded in the early-sown crop (1,276.7 kg ha⁻¹) and in ICCV 90201 (1,799.3 kg ha⁻¹), respectively. The yields were greatly reduced in all the cultivars during 2004–2005 and the highest yield was recorded in ICCX 810800 (524.7 kg ha⁻¹). Integrated disease management using resistant cultivars, optimum sowing period and foliar application of fungicides will improve chickpea production. The experiment under controlled environment and field conditions (during the epidemic year) showed a similar disease development.     

Characterization of Ascochyta isolates and susceptibility of pea cultivars to the ascochyta disease complex in Alberta

The relative virulence of 109 Ascochyta isolates collected from pea fields in Alberta from 1996 to 1998 were evaluated on 10-day-old seedlings by the excised leaf-assay technique. Twenty-eight isolates were avirulent, while the others produced lesions of various sizes on pea leaves. DNA samples from 86 isolates were amplified by the RAPD technique using PCR with single primers. One dominant genotype of Ascochyta pisi was identified throughout Alberta, but variations in virulence were not clearly differentiated by the RAPD technique. Five Ascochyta isolates, four virulent and one avirulent, were used to assess the susceptibility of 20 field pea cultivars available in Alberta, including 13 yellow types and seven green types. Based on symptom development, the yellow-type cultivars Swing, Eiffel and Delta, and the green-type cultivar Orb, were the most susceptible. Of yellow-type cultivars, Voyageur, Carneval and Montana were most resistant to A scochyta infection.     

Identification of the mutation responsible for resistance to QoI fungicides and its detection in Ascochyta rabiei (teleomorph Didymella rabiei)

This study characterized a fragment of the cytochrome b gene from Ascochyta rabiei isolates collected in North Dakota, USA, that varied in sensitivity to quinone‐outside inhibitor (QoI) fungicides. The sequenced genomic DNA fragment contained a group I intron immediately after codon 131. The size of the cytochrome b gene was estimated to be over 4·6 kb. Multiple alignment analysis of cDNA and protein sequences revealed a mutation that changed the codon for amino acid 143 from GGT to GCT, introducing an amino acid substitution from glycine to alanine (G143A), which is frequently associated with QoI resistance. Based on this mutation, a diagnostic PCR assay was developed using an approach called mismatch amplification mutation assay. This method was successfully validated by testing a total of 70 A. rabiei isolates, of which 38 isolates were found to be QoI‐resistant. This fast and accurate PCR assay provides a very useful and simple screening method for QoI resistance in A. rabiei isolates.     

大麦条纹病菌致病性分析及抗性种质精细鉴定

为探明大麦条纹病菌Pyrenophora graminea的致病性差异,鉴定筛选抗病大麦种质材料,将15株大麦条纹病菌菌株通过三明治法种子幼芽接种至5个鉴别品种,分析菌株的致病性分化情况;通过在田间孢子喷雾穗部接种和三明治法种子幼芽接种对145个田间种植表现抗病品种进行抗条纹病精细鉴定。结果显示,15株菌株对5个鉴别品种致病性有一定差异,可划分为9个致病类型,其中致病类型Ⅱ、Ⅲ和Ⅳ为优势致病类型,每个致病类型菌株数均占总菌株数的20.0%,其它致病类型的菌株均占总菌株数的6.7%;致病类型Ⅰ菌株致病力最强,对5个鉴别品种表现高感。孢子悬浮液喷雾接种田间试验结果显示,90个大麦品种表现为抗病,占总数的62.1%,其中表现免疫的品种12个、高抗品种26个、中抗品种52个,分别占总数的8.3%、17.9%、35.9%。种子幼芽接种温室条件下发病鉴定结果显示,90个抗病品种接种致病类型ⅢP05菌株,对其表现抗病的品种有28个,占供试品种总数的31.1%,表现免疫的品种10个、高抗品种7个、中抗品种11个,分别占供试品种总数的11.1%、7.8%、12.2%;接种致病类型ⅠP01菌株,对其表现抗...     

云南省水稻细菌性条斑病菌的致病型划分和水稻抗性资源的鉴定

 为明确云南省水稻细菌性条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)的致病力分化以及不同类型水稻品种对Xoc的抗感特性,通过针刺接种法将云南省8个稻区采集的86株Xoc菌株,接种于6个携带不同抗性基因的水稻鉴别品种(IRBB4、IRBB5、IRBB14、IRBB18、IRBB21和IR24)。根据这些菌株在鉴别品种上的毒力差异进行了UPGMA聚类分析,将其划分为9个致病型(Ⅰ型 ~ Ⅸ 型)。其中,Ⅰ型为优势菌群,分布频率为50.5%。对不同稻区的优势菌群进行分析,发现云南省各稻区Xoc的致病型呈多样性分布,以强毒力的Ⅰ型为高频率致病型。选用 Ⅰ 型、Ⅱ 型和 Ⅵ 型代表菌株对云南省的80个主栽和区试水稻品种进行抗性评价,对3个致病型表现抗性的材料比例分别为30.0%、35.0%和57.5%。筛选出9个对3种致病型都表现为抗性的品种,其中“Deyou16”和“Changgui2”表现为高抗。研究结果可为云南省防治水稻细菌性条斑病的水稻区域性布局和抗性品种的利用提供理论依据。     

Validation of a QTL for resistance to ascochyta blight linked to resistance to fusarium wilt race 5 in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Ascochyta blight caused by Ascochyta rabiei and fusarium wilt caused by Fusarium oxysporum. f. sp. ciceris are the two most serious diseases of chickpea (Cicer arietinum). Quantitative trait loci (QTL) or genes for ascochyta blight resistance and a cluster of resistance genes for several fusarium wilt races (foc1, foc3, foc4 and foc5) located on LG2 of the chickpea map have been reported independently. In order to validate these results and study the linkage relationship between the loci that confer resistance to blight and wilt, an intraspecific chickpea recombinant inbred lines (RIL) population that segregates for resistance to both diseases was studied. A new LG2 was established using sequence tagged microsatellite sites (STMS) markers selected from other chickpea maps. Resistance to race 5 of F. oxysporum (foc5) was inherited as a single gene and mapped to LG2, flanked by the STMS markers TA110 (6.5 cM apart) and TA59 (8.9 cM apart). A QTL for resistance to ascochyta blight (QTL_AR3) was also detected on LG2 using evaluation data obtained separately in two cropping seasons. This genomic region, where QTL_AR3 is located, was highly saturated with STMS markers. STMS TA194 appeared tightly linked to QTL_AR3 and was flanked by the STMS markers TR58 and TS82 (6.5 cM apart). The genetic distance between foc5 and QTL_AR3 peak was around 24 cM including six markers within this interval. The markers linked to both loci could facilitate the pyramiding of resistance genes for both diseases through MAS.     

水旱两种栽培模式下海南山栏稻对白叶枯病抗性鉴定与评价

为研究山栏稻对白叶枯病抗性,本试验以17个山栏稻品种为材料,在大田与温室大棚中同时种植,于大田中观察山栏稻在水、旱两种栽培模式下对海口当地白叶枯病菌的自然抗性并测产;在温室中检测水、旱两种栽培模式下山栏稻对菲律宾白叶枯病菌小种PXO99(P6)、PX145(P7)的抗性;并以基因功能性标记检测其抗性基因,分析各指标与抗病性的关系。结果表明,大田环境下17个供试品种中有12个品种在水作栽培模式下感病指数高于旱作,有10个品种水作栽培模式下产量比旱作高。温室中用P6接种山栏稻,水作模式下仅有1个品种表现抗病,8个品种感病;旱作模式下2个品种表现抗病,3个品种感病。接种P7,水作时表现抗病和感病的品种数量分别为7个和2个;旱作时表现抗病的品种高达9个,仅1个品种感病。供试品种中有16个品种含有Xa1抗病基因,4个品种含有Xa27抗病基因,所有品种均不含xa13、Xa21抗病基因。因此认为,山栏稻对海口当地白叶枯病菌小种以及菲律宾白叶枯病菌小种都有一定抗性,且抗性受栽培条件的影响,山栏稻旱作时抗性强于水作。     

A simple detached leaf assay provides rapid and inexpensive determination of pathogenicity of Pythium isolates to ‘all year round’ (AYR) chrysanthemum roots

A detached leaf assay was developed to determine the pathogenicity of Pythium isolates to cut‐flower chrysanthemum roots. Leaves from young plants were excised and inoculated by insertion of a plug of mycelium into a slit cut in the excised petiole. After incubation leaves were assessed for presence and extent of necrosis. Necrosis indicated pathogenicity and was consistently confirmed by comparisons with whole plant inoculations. The rate of necrosis spread also gave some indication of virulence. Isolates of Pythium sylvaticum, P. ultimum and HS group were the most virulent, with a mean rate of spread of 14·6 mm per day, significantly (P < 0·05) faster than the mean rate of spread, 1·6 mm per day, of less virulent isolates. Less virulent isolates included P. irregulare, P. oligandrum and P. aphanidermatum. The latter was unexpected, as P. aphanidermatum is an important species in pythium root rot epidemics in chrysanthemums elsewhere. The value of the detached leaf assay for screening large numbers of isolates was demonstrated in a survey of isolates from clinic samples from chrysanthemum nurseries and in a series of dilution‐plating experiments looking at numbers of Pythium propagules in commercial chrysanthemum beds showing root rot. In the survey, the predominant pathogenic species was identified as P. sylvaticum and the most likely source of infection was contaminated soil as opposed to blocking media or irrigation water, whilst in soil colonization studies the use of detached leaf assays demonstrated a relationship between pathogenic inoculum concentration in soil and the expression of root rot symptoms.     

25份北欧小麦抗白粉性评价及抗白粉病基因标记鉴定

采用苗期人工接菌的方法鉴定了25份北欧小麦对河南省7个不同地区小麦白粉病菌分离物的抗性,同时利用与已知抗白粉病基因Pm2、Pm3f、Pm4、Pm6、Pm13、Pm16、Pm21和Pm24连锁的分子标记对其中抗性优良的材料进行基因检测。结果表明,8份材料对所有接种的白粉病菌菌株表现高抗或免疫;其中NGB7476和NGB7809携带Pm4a和Pm16基因, NGB7481和NGB9954携带Pm6和Pm16基因,NGB9955和NGB9956同时携带Pm4a、Pm6和Pm16基因,8份材料均不携带Pm2、Pm3f、Pm4b、Pm13、Pm21 和Pm24基因。     

A SNaPshot assay for the rapid and simple detection of known point mutations conferring resistance to ACCase‐inhibiting herbicides in Lolium spp.

Evolution of resistance to herbicides in weeds is becoming an increasing problem worldwide. To develop effective strategies for weed control, a thorough knowledge of the basis of resistance is required. Although non‐target‐site‐based resistance is widespread, target site resistance, often caused by a single nucleotide change in the gene encoding the target enzyme, is also a common factor affecting the efficacies of key herbicides. Therefore, fast and relatively simple high‐throughput screening methods to detect target site resistance mutations will represent important tools for monitoring the distribution and evolution of resistant alleles within weed populations. Here, we present a simple and quick method that can be used to simultaneously screen for up to 10 mutations from several target site resistance‐associated codons in a single reaction. As a proof of concept, this SNaPshot multiplex method was successfully applied to the genotyping of nine variable nucleotide positions in the CT domain of the chloroplastic ACCase gene from Lolium multiflorum plants from 54 populations. A total of 10 nucleotide substitutions at seven of these nine positions (namely codons 1781, 1999, 2027, 2041 2078, 2088 and 2096) are known to confer resistance to ACCase‐inhibiting herbicides. This assay has several advantages when compared with other methods currently in use in weed science. It can discriminate between different nucleotide changes at a single locus, as well as screening for SNPs from different target sites by pooling multiple PCR products within a single reaction. The method is scalable, allowing reactions to be carried out in either 96‐ or 384‐well plate formats, thus reducing work time and cost.