Resistance to ascochyta blights of cool season food legumes

Ascochyta blights are the most important diseases of cool season food legumes (peas, lentils, chickpeas, and faba beans) and are found in nearly all production regions. Despite having the same common disease name, the pathogen species differ for each of the crops. These diseases cause serious yield losses under favourable cool and humid conditions. Planting resistant cultivars is often the first choice and most economical means in managing the diseases. Therefore breeding for resistance to ascochyta blights has been an important objective of many cool season food legume research programmes. Systematic screening of germplasm collections at international research centres and other national research programmes have identified useful resistance sources that have been used successfully to breed resistant or tolerant cultivars. Genetic studies have revealed inheritance patterns of the resistance genes. Genetic linkage analyses and QTL mapping have identified molecular markers that could be useful for marker-assisted selection and gene pyramiding. In general, research towards developing resistance to ascochyta blights in cool season food legume faces mainly two limitations: the lack of availability of efficient resistance sources and the lack of a good understanding of the variability of the pathogen populations. Research efforts to alleviate these limitations should be pursued. Given that modern technologies of marker development and genomics are available, further advances in deploying resistance to manage ascochyta blights in this group of legume crops will depend on concerted efforts in developing accurate screening procedures with adequate knowledge of pathogen variability and identification of additional sources of resistance.     

Biotic factors affecting the expression of partial resistance in pea to ascochyta blight in a detached stipule assay

The expression of partial resistance in pea to ascochyta blight (caused by Mycosphaerella pinodes) was studied in a detached stipule assay by quantifying two resistance components (fleck coalescence and lesion expansion) using the method of point inoculation of stipules. Factors determining optimal conditions for the observation of partial resistance are spore concentration, the age of the fungal culture prior to spore harvest and the pathogenicity of the isolate used for testing. Partial resistance was not expressed when spore concentration was high or when the selected isolate was aggressive. Furthermore, assessments of components of partial resistance were highly correlated with disease severity in a seedling test. A screening protocol was developed based on inoculations of detached stipules to study partial resistance in pea. To simplify the rating process, a more comprehensive disease rating scale which took into account fleck coalescence and lesion expansion was tested by screening a large number of genotypes.     

Integrated disease management of ascochyta blight in pulse crops

Ascochyta blight causes significant yield loss in pulse crops worldwide. Integrated disease management is essential to take advantage of cultivars with partial resistance to this disease. The most effective practices, established by decades of research, use a combination of disease-free seed, destruction or avoidance of inoculum sources, manipulation of sowing dates, seed and foliar fungicides, and cultivars with improved resistance. An understanding of the pathosystems and the inter-relationship between host, pathogen and the environment is essential to be able to make correct decisions for disease control without compromising the agronomic or economic ideal. For individual pathosystems, some components of the integrated management principles may need to be given greater consideration than others. For instance, destruction of infested residue may be incompatible with no or minimum tillage practices, or rotation intervals may need to be extended in environments that slow the speed of residue decomposition. For ascochyta-susceptible chickpeas the use of disease-free seed, or seed treatments, is crucial as seed-borne infection is highly effective as primary inoculum and epidemics develop rapidly from foci in favourable conditions. Implemented fungicide strategies differ according to cultivar resistance and the control efficacy of fungicides, and the effectiveness of genetic resistance varies according to seasonal conditions. Studies are being undertaken to develop advanced decision support tools to assist growers in making more informed decisions regarding fungicide and agronomic practices for disease control.     

Diagnostics,genetic diversity and pathogenic variation of ascochyta blight of cool season food and feed legumes

Molecular diagnostic techniques have been developed to differentiate the Ascochyta pathogens that infect cool season food and feed legumes, as well as to improve the sensitivity of detecting latent infection in plant tissues. A seed sampling technique was developed to detect a 1% level of infection by Ascochyta rabiei in commercial chickpea seed. The Ascochyta pathogens were shown to be genetically diverse in countries where the pathogen and host have coexisted for a long time. However, where the pathogen was recently introduced, such as A. rabiei to Australia, the level of diversity remained relatively low, even as the pathogen spread to all chickpea-growing areas. Pathogenic variability of A. rabiei and Ascochyta pinodes pathogens in chickpea and field pea respectively, appears to be quantitative, where measures of disease severity were based on aggressiveness (quantitative level of infection) rather than on true qualitative virulence. In contrast, qualitative differences in pathogenicity in lentil and faba bean genotypes indicated the existence of pathotypes of Ascochyta lentis and Ascochyta fabae. Therefore, reports of pathotype discrimination based on quantitative differences in pathogenicity in a set of specific genotypes is questionable for several of the ascochyta-legume pathosystems such as A. rabiei and A. pinodes. This is not surprising since host resistance to these pathogens has been reported to be mainly quantitative, making it difficult for the pathogen to overcome specific resistance genes and form pathotypes. For robust pathogenicity assessment, there needs to be consistency in selection of differential host genotypes, screening conditions and disease evaluation techniques for each of the Ascochyta sp. in legume-growing countries throughout the world. Nevertheless, knowledge of pathotype diversity and aggressiveness within populations is important in the selection of resistant genotypes.     

Control of seed-borne pathogens on legumes by microbial and other alternative seed treatments

Greenhouse trials were carried out in order to test the efficacy of different seed treatments as alternatives to chemicals against Colletotrichum lindemuthianum cause of anthracnose on bean and Ascochyta spp. cause of Ascochyta blights on pea, respectively. Resistance inducers, commercially formulated microorganisms, non-formulated selected strains of different microorganisms (fungi, bacteria and yeasts) and plant extracts were applied as dry or liquid seed treatments on naturally infested seeds. Seedling emergence and disease incidence and/or severity were recorded. Almost all seed treatments turned out to be ineffective in controlling the Ascochyta infections, which is in line with the literature stating that these pathogens are difficult to control. The only alternative treatments that gave some control of Ascochyta spp. were thyme oil and a strain of Clonostachys rosea. The resistance inducers tested successfully controlled infections of bean by C. lindemuthianum. Among the formulated microorganisms, Bacillus subtilis-based formulations provided the best protection from anthracnose. Some strains of Pseudomonas putida, a disease-suppressive, saprophytic strain of Fusarium oxysporum and the mustard powder-based product Tillecur also proved to be effective against bean anthracnose. However, among the resistance inducers as well as among the other groups, certain agents caused a significant reduction of plant emergence. Different alternative seed treatments can therefore be used for the control of C. lindemuthianum on bean, while on pea only thyme oil and a strain of Clonostachys rosea showed some effectiveness against Ascochyta spp.     

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.     

Improvement and validation of a pea crop growth model to simulate the growth of cultivars infected with Ascochyta blight (<Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis>)

A model simulating the growth of a pea crop infected with Ascochyta blight was improved and validated using 6 spring pea cultivars, all equally susceptible to Ascochyta blight, but differing in architectural features (stem height, branching ability, standing ability). This model takes into account the spatial distribution of the disease, including the contribution of each layer of the canopy to the radiation interception efficiency (RIE) and the radiation use efficiency (RUE) of the crop. The decreasing contribution of each layer due to the disease was estimated by the relationship between the photosynthesis of a layer and its disease score. The effect of disease on photosynthesis was assessed in controlled conditions as a means of evaluating the effect of disease on each cultivar. All cultivars were affected equally. In field conditions, cultivars with different canopy architectures displayed differences in the profile of disease on leaves. Cultivar Aladin reached higher disease levels at the top of the plant. Epidemics affected crop growth, and the cultivars tested differed in the magnitude of the decrease in growth. Observed and simulated data were compared. The disease-coupled crop growth model gave satisfactory predictions of crop growth for the six cultivars tested.     

Identification of genes expressed during spore germination of Mycosphaerella pinodes

Mycosphaerella blight, caused by Mycosphaerella pinodes, is one of the major diseases of cultivated pea (Pisum sativum L.). To isolate the genes that are up- and down-regulated during spore germination, suppression subtraction hybridization (SSH) was performed between ungerminated and germinated spores. The 232 and 128 clones from forward and reverse libraries, respectively, were collected, sequenced, and analyzed with a BLASTX homology search. About 95% of the 32 selected clones were expressed during spore germination on a paper sheet and during infection of pea leaves. We discuss the applicability of the SSH libraries for analyzing M. pinodes genes involved in the early stage of infection.     

Localization and responsiveness of a cowpea apyrase VsNTPase1 to phytopathogenic microorganisms

Apyrases (NTPases) are associated with both compatible and incompatible interactions between plants and microorganisms. Previously we reported that the ATPase activities of cell-wall-bound apyrases of several leguminous plants, such as pea, cowpea, soybean, and kidney bean, were enhanced by a glycoprotein elicitor and were inhibited in a species-specific manner by mucin-type glycopeptide suppressors secreted from a pea pathogenic fungus, Mycosphaerella pinodes. In this study, we isolated two apyrase genes, VsNTPase1 and VsNTPase2, from a cDNA library of Vigna sinensis Endl. cv. Sanjakusasage. Based on phylogenetic analysis, VsNTPase1 may belong to a group that responds to environmental stimuli. In a transient assay using DNA bombardment, a fusion protein of green fluorescent protein (GFP) and the N-terminal putative signal sequence of VsNTPase1 was distributed in the nucleus, cytoplasm (cytoskeletal structure), and cell wall. On the other hand, a fusion protein of GFP and the N-terminal putative VsNTPase2-signal sequence was localized in the cytoplasm, especially in small particles (perhaps mitochondria). A recombinant VsNTPase1 expressed in Spodoptera frugiperda 21 cells responded directly to signal molecules from several phytopathogenic microorganisms. Here, we discuss the role of apyrases in recognizing and responding to exogenous signals. The nucleotide sequences of VsNTPase1 and VsNTPase2 in this article have been submitted to DDBJ as accession numbers AB196769 and AB196770, respectively.     

Relation between cropping frequency of peas and other legumes and foot and root rot in peas

The relation between the frequency of legume crops in a rotation and the root rot severity in pea was examined in a field survey. Additionally, greenhouse experiments were performed with soil samples from legume rotation trials or from farmers' fields. The frequency of pea crops in current rotations proved to be much less than the recommended value of one in six years. The correlation between pea root rot and the number of years that pea or other legumes were not grown on the field under consideration (called crop interval) was weak. Root rot severity correlated better with the frequency of peas or legumes in general over a period of 18 years, but the frequency still explained only a minor fraction of the variation in disease index. Some experimental data pointed to the occurrence of a highly specific pathogen microflora with continuous cropping of only one legume species, but this phenomenon probably does not occur in farmers' fields. In field samples, root disease index for pea correlated well with that for field bean. The survival of resting structures of pathogens such asAphanomyces euteiches probably explains why the frequency of legume cropping has a higher impact than crop interval on root disease incidence. Pea-free periods and legume frequencies have a poor predictive value for crop management purposes.     

Influence of humidity on incidence of Didymella bryoniae on cucumber leaves and growing tips under controlled environmental conditions

The influence of relative humidity, leaf wetting, mechanical injury and inoculum concentration on the incidence ofDidymella bryoniae on growing tips and young and older leaves of cucumber was studied in growth chambers.Infection was rare at 60% r.h. It increased at 95% r.h. and was most serious if the leaves were kept wet. A period of 1 hour of free water was sufficient for the initial stage of infection. For further expansion of the disease, leaf wetness was required.A high relative humidity did not predispose leaves to infection byD. bryoniae. Wounding was essential for infection of older leaves, but not for infection of young plant tissue.A higher conidial concentration increased infection. Without keeping, the leaves wet at 95% r.h. a tenfold conidial concentration was needed to get equal infection as with leaf wetting.To control the disease by means of the climate, it is of major importance to prevent the presence of free water on plant parts.Samenvatting De invloed van de relatieve luchtvochtigheid, het bevochtigen van het blad, mechanische beschadiging en inoculumconcentratie op het optreden vanDidymella bryoniae op groeipunten en jonge en oudere bladeren van komkommer is in klimaatkasten onderzocht.Aantasting kwam zelden voor bij 60% R.V., nam toe bij 95% R.V. en was het ernstigst als de bladeren nat werden gehouden. Voor de eerste fase van infective was de aanwezigheid van vrij water gedurende 1 à 2 uur voldoende. Voor een verdere uitbreiding van de aantasting moest het blad nat zijn.Een hoge relatieve luchtvochtigheid had geen predispositie-effect of de infectie van bladeren doorD. bryoniae.Voor de infectie van oudere bladeren was verwonding nodig, voor die van jong planteweefsel niet.Een hogere concentratie van conidiën verhoogde de aantasting. Zonder het blad nat te houden, was een tienvoudige concentratie van conidiën nodig om een gelijke infectie te verkrijgen als met bladbevochtiging.Voor de bestrijding van de ziekte via het klimaat is het tegengaan van de aanwezigheid van vrij water op plantedelen van het grootste belang.Seconded to the Glasshouse Crops Research and Experiment Station, Zuidweg 38, 2671 MN Naaldwijk, the Netherlands.     

Comparison of the epidemiology of Metopolophium dirhodum and Sitobion avenae on winter wheat

The epidemiology ofMetopolophium dirhodum and its natural enemies on winter wheat was studied from 1975 to 1979 inclusive. AlateM. dirhodum colonize wheat from the middle of May onwards. Immigration occurs until mid-June. Population growth rates ofM. dirhodum can be as high as or occasionally even higher than those ofSitobion avenae. InM. dirhodum alatae are formed in a high proportion throughout the epidemic whereas inS. avenae the % of alatae becomes high at the end. In both species most of these alatae seem to emigrate. Both species have a similar potential to become a pest.Samenvatting De epidemiologie vanMetopolophium dirhodum werd bestudeerd van 1975–1989 en vergeleken met die vanSitobion avenae. Beide soorten migreren naar tarwe in de loop van mei. De relatieve populatie groeisnelheid vanM. dirhodum bleek tijdelijk zeer hoog te zijn (verdubbelingstijd 1,7 dag tegenover 2,0 voorS. avenae). Over de gehele periode van de epidemie is er weinig verschil in dagelijkse relatieve groeisnelheid van de populatie.Bij de ineenstorting, die altijd begint voor het deegrijpstadium van de tarwe, is van belang het ontstaan van gevleugelde bladluizen. Deze verlaten merendeels het gewas. Het belang van de natuurlijke vijanden voor de afname van de populatie is niet altijd duidelijk maar is soms groot en veroorzaakt dan een vroege ineenstorting van de plaag. M. dirhodum heeft even grote mogelijkheden de plaagstatus te bereiken alsS. avenae maar door zijn soms tijdelijk groter vermeerderingsvermogen zal het voorspellen moeilijker zijn.     

Host status and reaction of <Emphasis Type="Italic">Medicago truncatula</Emphasis> accessions to infection by three major pathogens of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>)

Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches.     

A binding protein for fungal signal molecules in the cell wall of Pisum sativum

In the plant cell wall of Pisum sativum seedlings, we found an NTPase (E.C. 3.6.1.5.) with ATP-hydrolyzing activity that was regulated by an elicitor and suppressors of defense from pea pathogen Mycosphaerella pinodes. The ATPase-rich fraction was purified from pea cell walls by NaCl solubilization, ammonium sulfate precipitation, and chromatography with an ATP-conjugated agarose column and an anion-exchange column. The specific activity of the final ATPase-rich fraction increased 600-fold over that of the initial NaCl-solubilized fraction. The purified ATPase-rich fraction also had peroxidase activity and generated superoxide, both of which were regulated by the M. pinodes elicitor and suppressor (supprescins). Active staining and Western blot analysis also showed that the ATPase was copurified along with peroxidases. In this fraction, a biotinylated elicitor and the supprescins were bound primarily and specifically to ca. 55-kDa protein (CWP-55) with an N-terminal amino acid sequence of QEEISSYAVVFDA. The cDNA clone of CWP-55 contained five ACR domains, which are conserved in the apyrases (NTPases), and the protein is identical to a pea NTPase cDNA (GenBank accession AB071369). Based on these results, we discuss a role for the plant cell wall in recognizing exogenous signal molecules.     

Influence of time of transition from night to day temperature regimes on incidence of Didymella bryoniae and influence of the disease on growth and yield of glasshouse cucumbers

The influence of transition from night to day temperature 3 h before, 1 h before, 1h after and 3 h after sunrise on the incidence ofDidymella bryoniae was studied both on inoculated and on uninoculated glasshouse-grown cucumber plants. The effect of inoculation on plant growth and fruit production was studied as well.The later the transition to day temperature took place, the longer were the periods with a high relative air humidity and of condensation of water on fruits.The time of transition had no effect on plant growth, yield, disease incidence on growing tips, number of lesions on the main stems of uninoculated plants and external fruit rot. The later the transition to day temperature took place, the more lesions on the main stem of inoculated plants appeared and the higher was the incidence of internal fruit rot.Inoculation of plants increased the number of lesions on the main stem, the disease incidence on growing tips, the production of misshapen fruits and the internal and external fruit rot. The number of secondary side shoots was incrreased but the total number of their internodes was reduced by inoculation.Inoculation caused an 18% reduction in number of internodes over a period of four weeks and a 10% reduction in number of fruits in the corresponding harvest period.The consequences of a more humid glasshouse climate and of a high infection pressure ofD. bryoniae for the grower are briefly discussed.Samenvatting De invloed van het 3 uur vóór, 1 urr vóór, 1 uur na en 3 uur na zonsopgang overgaan van de nacht-naar de dagtemperatuur op het optreden vanDidymella bryoniae werd zowel op geïnoculleerde als op niet-geïnocullerde planten van kaskomkommers onderzocht. De invloed van inoculatie op de groei van de planten en de produktie van vruchten werd eveneens nagegaan.Hoe later naar de dagtemperatuur werd overgegaan, hoe langer de perioden met een hoge relatieve luchtvochtigheid waren en hoe langer de perioden waarin condensatie van water op vruchten optrad.Het tijdstip van overgang had geen effect op de groei van de planten, de opbrengst, de aantasting van groeipunten, het aantal lesies op de hoofdstengel van niet geïnoculeerde planten en uitwendig vruchtrot. Hoe later naar de dagtemperatuur werd overgegaan, hoe meer lesies na vier weken op de hoofdstengel van geïnoculeerde planten en hoe meer vruchten met inwendig rot voorkwamen.Door inoculatie van de planten nam het aantal lesies op de hoofdstengel, de aantasting van groeipunten, de produktie van stekvruchten en het aantal vruchten met inen uitwendig rot toe. Het aantal zijscheuten van de tweede orde nam toe, maar het totaal aantal internodiën ervan nam door inoculatie af. Inoculatie reduceerde het aantal internodiën met 18% over een periode van vier weken en die van het aantal vruchten met 10% in de overeenkomstige oogstperiode. De praktische consequenties van eenSeconded to the Glashouse Crops Research and Experiment Station, Zuidweg 38, 2671 MN Naaldwijk, the Netherlands     

The role of seedling infection in epiphytotics of ascochyta blight on chickpea

Didymella rabiei, the causal agent of ascochyta blight, survives on infected seeds and seedlings. Diseased seedlings originating from infected seeds occasionally serve as the source for primary infection in chickpea crops. Experiments carried out independently in Australia and in Israel provided quantitative information on the temporal and spatial distribution of ascochyta blight from initial infections and on the relationship between the amount of initial infection and the intensity of subsequent epiphytotics for cultivars differing in susceptibility to the pathogen. Disease spread over short distances (<10 m) from individual primary infections, was governed by rain and wind, and was up to five times greater down-wind than up-wind. Cultivar response to D. rabiei significantly affected the distance and area over which disease spread and the intensity of the disease on infected plants. At onset of the epiphytotic, the relationship between disease spread and time was exponential (P < 0.05; R ² > 0.95) and the area of the resulting foci was over 10 times greater in susceptible cultivars than in resistant cultivars. Regression equations showed the relationship between disease severity and the distance from the focus-plants was inverse-linear for all cultivars tested (P < 0.05). A simulation model based on the experimental data revealed that even if primary infection is infrequent (less than 1% of plants), the consequences are potentially devastating when susceptible cultivars are used. The epidemiological information and simulation model generated by this study provide an increased understanding of the development of an epiphytotic in which the primary foci of disease originate from infected chickpea seedlings.     

Effects of temperature increase on the epidemiology of three major vector-borne viruses

The epidemiologies of Maize streak virus (MSV), Maize stripe virus (MSpV), and Maize mosaic virus (MMV) were compared in La Réunion over a three year-period. Disease incidence caused by each virus was assessed, and the leaf and planthopper vector populations (Cicadulina mbila and Peregrinus maidis) were estimated in weekly sowings of the temperate, virus-susceptible maize hybrid INRA 508 and of the composite resistant cv. IRAT 297. MSV caused the most prevalent disease and MMV the least, with lower incidences in cv. IRAT 297 than in INRA 508. For each plant–virus–vector combination, (a) disease incidence was positively correlated to vector abundance, often with 1 month of time lag; (b) annual periodicity of disease incidence and of vector numbers was consistent with highest autocorrelations and a time lag of 12 months, (c) vector numbers and disease incidence were closely associated with temperature fluctuations, both remaining relatively constant below 24°C and increasing rapidly above this threshold temperature. By contrast, relationships with rainfall and relative humidity (RH) were less consistent. Overall, 63 to 80% of the variance of disease incidence was explained through stepwise regression with vector number, temperature, and sometimes also rainfall or RH. The simple epidemiological model proposed underlines the close link between increased temperature and possible (re-) emergence of these three diseases in a maize cropping area.     

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.     

烟草叶斑病病原菌的鉴定及其生物学特性

为明确贵州省新发现的烟草叶斑病病原菌种类和生物学特性,2019年自田间采集具有典型发病症状的烟叶,采用组织分离法对其进行分离纯化,对分离菌株进行致病性测定,观察病原菌形态学特征,基于ITS、LSU、tub2和rpb2基因序列进行分子生物学鉴定,同时对病原菌的生物学特性进行测定。结果显示,烟草叶斑病病斑呈圆形或椭圆形,病斑中间呈浅褐色,边缘呈棕色,周围环绕黄色晕圈。经分离纯化共获得3株病原菌,形成的菌落为深褐色,气生菌丝呈白色;分子孢子器呈球形或扁球形,为深褐色,大小为79.3～91.7μm×148.4～167.3μm;分生孢子呈椭圆形,光滑,无隔膜,具0～3个油球,大小为3.5～5.6μm×1.7～2.9μm。根据形态学特征和分子生物学特征将其鉴定为Didymella segeticola。该病原菌菌丝生长适宜温度为20～25℃;适宜pH为6～10,最适碳源为乳糖,最适氮源为蛋白胨,通气和光暗交替环境有利于其生长。