Leptosphaeria Maculans Research Articles

Pydiflumetofen: An SDHI seed‐applied fungicide, a potential tool for the canola‐blackleg management toolbox

AbstractBlackleg is a major fungal disease of canola caused by Leptosphaeria maculans. Due to the breakdown of current host resistance, having an effective fungicide to avoid seedling infection is important to avoid disease. Pydiflumetofen is a newly developed broad‐spectrum seed treatment fungicide in the succinate dehydrogenase inhibitor (SDHI) group. We evaluated the separate and combined application of pydiflumetofen and Vibrance Flexi on early‐season control of blackleg using moderately resistant canola cultivar SY4135 and susceptible cultivar Westar. In the greenhouse, seedlings inoculated with green fluorescent protein (GFP)‐tagged L. maculans showed lesion development and apoplastic mycelial growth in cotyledons in control and Vibrance Flexi treatments but no lesion development or in planta mycelial growth was observed in pydiflumetofen‐treated seedlings, in Westar or SY4135, 14 days postinoculation. SY4135 and Westar plants with pydiflumetofen‐treated seeds inoculated with L. maculans isolate D3 had significantly lower disease severity. Both host genotypes treated with pydiflumetofen showed lower disease severity estimates than the other treatments. Statistically significant disease severity reduction was cultivar dependent in the field. Canola is most susceptible to blackleg as a seedling, from the cotyledon to two‐ to four‐leaf stage. Pydiflumetofen protects the plant at the critical window for infection. Therefore, pydiflumetofen could be a potential tool that farmers can add to their blackleg management toolbox.

Genome-Wide Identification and Expression Analysis of nsLTP Gene Family in Rapeseed (Brassica napus) Reveals Their Critical Roles in Biotic and Abiotic Stress Responses.

Non-specific lipid transfer proteins (nsLTPs) are small cysteine-rich basic proteins which play essential roles in plant growth, development and abiotic/biotic stress response. However, there is limited information about the nsLTP gene (BnLTP) family in rapeseed (Brassica napus). In this study, 283 BnLTP genes were identified in rapeseed, which were distributed randomly in 19 chromosomes of rapeseed. Phylogenetic analysis showed that BnLTP proteins were divided into seven groups. Exon/intron structure and MEME motifs both remained highly conserved in each BnLTP group. Segmental duplication and hybridization of rapeseed’s two sub-genomes mainly contributed to the expansion of the BnLTP gene family. Various potential cis-elements that respond to plant growth, development, biotic/abiotic stresses, and phytohormone signals existed in BnLTP gene promoters. Transcriptome analysis showed that BnLTP genes were expressed in various tissues/organs with different levels and were also involved in the response to heat, drought, NaCl, cold, IAA and ABA stresses, as well as the treatment of fungal pathogens (Sclerotinia sclerotiorum and Leptosphaeria maculans). The qRT-PCR assay validated the results of RNA-seq expression analysis of two top Sclerotinia-responsive BnLTP genes, BnLTP129 and BnLTP161. Moreover, batches of BnLTPs might be regulated by BnTT1 and BnbZIP67 to play roles in the development, metabolism or adaptability of the seed coat and embryo in rapeseed. This work provides an important basis for further functional study of the BnLTP genes in rapeseed quality improvement and stress resistance.

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins.

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9-mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3.

In silico integration of disease resistance QTL, genes and markers with the Brassica juncea physical map

Brassica juncea (AABB), Indian mustard, is a source of disease resistance genes for a wide range of pathogens. The availability of reference genome sequences for B. juncea has made it possible to characterise the genomic structure and distribution of these disease resistance genes. Potentially functional disease resistance genes can be identified by co-localization with genetically mapped disease resistance quantitative trait loci (QTL). Here we identify and characterise disease resistance gene analogs (RGAs), including nucleotide-binding site–leucine-rich repeat (NLR), receptor-like kinase (RLK) and receptor-like protein (RLP) classes, and investigate their association with disease resistance QTL intervals. The molecular genetic marker sequences for four white rust (Albugo candida) disease resistance QTL, six blackleg (Leptosphaeria maculans) disease resistance QTL and BjCHI1, a gene cloned from B. juncea for hypocotyl rot disease, were extracted from previously published studies and used to compare with candidate RGAs. Our results highlight the complications for the identification of functional resistance genes, including the duplicated appearance of genetic markers for several resistance loci, including Ac2(t), AcB1-A4.1, AcB1-A5.1, Rlm6 and PhR2 in both the A and B genomes, due to the presence of homoeologous regions. Furthermore, the white rust loci, Ac2(t) and AcB1-A4.1, mapped to the same position on chromosome A04 and may be different alleles of the same gene. Despite these challenges, a total of nine candidate genomic regions hosting 14 RLPs, 28 NLRs and 115 RLKs were identified. This study facilitates the mapping and cloning of functional resistance genes for applications in crop improvement programs.

Effect of Fusarium blight, Phoma rot, and Sclerotinia blight on rapeseed and mustard plant productivity

The article presents the research data on the effect of Fusarium blight, Phoma rot, and Sclerotinia blight on the plant productivity of winter and spring rapeseed, brown mustard, and white mustard. As a result of studies, we found that Fusarium blight (the pathogens are fungi of the genus Fusarium Link) was the most harmful for the spring rapeseed, brown and white mustard – affection by it significantly decreased plant productivity. At 4 points of affection degree of plants, the harmfulness of the disease reached 71.2, 82.4, and 87.2 %, respectively. On winter rapeseed and mustard, plant productivity was considerably decreased by affection by Phoma rot in the form of a stem cancer (the pathogen is a fungus Leptosphaeria maculans (Desm.) Ces. et. De Not) and the stem form of Sclerotinia blight (the pathogen is a fungus Sclerotinia sclerotiorum (Lib.) De Bary.). The harmfulness of these diseases at 4 points of affection degree of plants reached 56.7 % and 66.7 % on rapeseed, and 56.6 % and 70.7 % on mustard, respectively.

Genetic structure and phylogenetic relationships of Leptosphaeria maculans and L. biglobosa in Northern regions of Iran

Blackleg, caused by Leptosphaeria maculans, and L. biglobosa, is a major disease that hinders canola (Brassica napus) production worldwide. In this study, 51 Leptosphaeria isolates, and previously deposited sequences were examined for genetic diversity using ITS, β-tubulin, tef and act gene regions. Phylogenetic analyses showed that the isolates fell within two sub-species of brassicae and canadensis. Out of 51 Iranian isolates, 55%, 37%, and 8% were found as L. biglobosa canadensis, L. biglobosa brassicae, and L. maculans, respectively. Accordingly, the canadensis sub-species was found to be the predominant sub-species in Iran. There was no difference between Iranian and foreign brassicae sub-species in terms of ITS nucleotide sequences. This paper confirms the presence of brassicae and canadensis sub-species in Iran and describes the distribution of L. biglobosa in the country. Moreover, Leptosphaeria species were distinguished using a phylogenetic tree based on the tef region.

Arabinogalactan Protein-Like Proteins From Ulva lactuca Activate Immune Responses and Plant Resistance in an Oilseed Crop.

Natural compounds isolated from macroalgae are promising, ecofriendly, and multifunctional bioinoculants, which have been tested and used in agriculture. Ulvans, for instance, one of the major polysaccharides present in Ulva spp. cell walls, have been tested for their plant growth-promoting properties as well as their ability to activate plant immune defense, on a large variety of crops. Recently, we have characterized for the first time an arabinogalactan protein-like (AGP-like) from Ulva lactuca, which exhibits several features associated to land plant AGPs. In land plant, AGPs were shown to play a role in several plant biological functions, including cell morphogenesis, reproduction, and plant-microbe interactions. Thus, isolated AGP-like proteins may be good candidates for either the plant growth-promoting properties or the activation of plant immune defense. Here, we have isolated an AGP-like enriched fraction from Ulva lactuca and we have evaluated its ability to (i) protect oilseed rape (Brassica napus) cotyledons against Leptosphaeria maculans, and (ii) its ability to activate immune responses. Preventive application of the Ulva AGP-like enriched fraction on oilseed rape, followed by cotyledon inoculation with the fungal hemibiotroph L. maculans, resulted in a major reduction of infection propagation. The noticed reduction correlated with an accumulation of H2O2 in treated cotyledons and with the activation of SA and ET signaling pathways in oilseed rape cotyledons. In parallel, an ulvan was also isolated from Ulva lactuca. Preventive application of ulvan also enhanced plant resistance against L. maculans. Surprisingly, reduction of infection severity was only observed at high concentration of ulvan. Here, no such significant changes in gene expression and H2O2 production were observed. Together, this study indicates that U. lactuca AGP-like glycoproteins exhibit promising elicitor activity and that plant eliciting properties of Ulva extract, might result not only from an ulvan-originated eliciting activities, but also AGP-like originated.

Molecular Interactions Between Leptosphaeria maculans and Brassica Species.

Canola is an important oilseed crop, providing food, feed, and fuel around the world. However, blackleg disease, caused by the ascomycete Leptosphaeria maculans, causes significant yield losses annually. With the recent advances in genomic technologies, the understanding of the Brassica napus-L. maculans interaction has rapidly increased, with numerous Avr and R genes cloned, setting this system up as a model organism for studying plant-pathogen associations. Although the B. napus-L. maculans interaction follows Flor's gene-for-gene hypothesis for qualitative resistance, it also puts some unique spins on the interaction. This review discusses the current status of the host-pathogen interaction and highlights some of the future gaps that need addressing moving forward.

Frequency and genetic variability of the avirulence gene AvrLm4-7 among Leptosphaeria maculans isolates collected in Oklahoma, USA

Blackleg, caused by Leptosphaeria maculans, is an important disease of canola managed primarily through host-plant resistance. Fungal avirulence (AvrLm) genes code for effectors recognized by major host-plant resistance (Rlm) genes. AvrLm4-7 is predominant in the pathogen population in Oklahoma and Kansas, USA, and interacts with the Rlm4 and Rlm7 resistance genes. High-resolution melting analysis and sequencing were used to evaluate the genetic variability of AvrLm4-7 in avirulent (A) and virulent (a) reference isolates, and in isolates (n=67) collected in Oklahoma from 2014 to 2017. Melting analysis of PCR products from the central primers (493 bp) and from new upstream external primers (105 bp) produced melting profiles that differed among avirulent (AvrLm4-AvrLm7 (A4A7)) and virulent (avrLm4-AvrLm7 (a4A7) and (avrLm4-avrLm7 (a4a7)) pathogen genotypes. Genotypic classification by melting analysis was supported by pathogenicity scores on seedling cotyledons of susceptible, Rlm4, and Rlm7 differentials. All of the local isolates were a4A7 genotypes. In the sequences of a4A7 reference isolates, all showed the expected mutation at bp 358 of C to G resulting in virulence on Rlm4, and silent mutations of either C to T at bp 256 or A to G at bp 134. These two haplotypes displayed unique melting profiles from the central primers in 64% and 36% of local isolates, respectively. Only the central primers produced pathotype-specific melting curves. Sustainable deployment of Rlm7 canola is needed for effective management of blackleg in the region. High-resolution melting will be useful to monitor allelic variability in AvrLm4-7 and for rapid detection of a4a7 strains.

Brassica napus genes Rlm4 and Rlm7, conferring resistance to Leptosphaeria maculans, are alleles of the Rlm9 wall-associated kinase-like resistance locus.

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Effect of wounding and wound age on infection of canola cotyledons by Leptosphaeria maculans, interacting with leaf wetness

Blackleg, caused by the fungus Leptosphaeria maculans, is an important disease of canola. Both ascospores and pycnidiospores of the fungus can infect intact leaves under conditions of extended leaf wetness, with infection then progressing further into the stem. In western Canada, spring is typically cool and dry and additional factors may be involved for successful infection. This study was designed to assess the effect of wounding and wound age on cotyledon infection, to evaluate the likelihood that flea beetle-feeding injuries could contribute to increased disease. Infection of canola cotyledons occurred readily via mechanical wounds in the absence of leaf wetness when seedlings were spray inoculated with pycnidiospores of L. maculans, whereas no infection occurred on intact cotyledons even under 6-h leaf wetness. Wound age also played a role in susceptibility; wounded tissues were less susceptible when plants were kept in a greenhouse for 8 h or longer before inoculation, with substantially reduced infection success relative to fresh wounds. This wound-age effect was similar on susceptible and resistant canola varieties. A high temperature (25°C) seemed to favour the healing of wounds, reducing susceptibility when compared with control (21/16°C) and low (10°C) temperatures. Leaf wetness post-wounding may hinder healing, and high post-inoculation humidity (80–90% relative humidity) increased infection via older wounds slightly, relative to lower (50–60%) relative humidity. These data provide a rationale for studying the effect of improved flea beetle control on early L. maculans infection of canola.

Molecular Markers for Identifying Resistance Genes in Brassica napus

Blackleg disease, caused by the fungal pathogen Leptosphaeria maculans, is the most devastating disease of canola (Brassica napus, oilseed rape) worldwide. Breeding for genetic resistance is the most widely used tool for controlling this disease and minimizing the impact on yield. To date, five resistance genes (Rlm2, LepR3, Rlm4, Rlm7, Rlm9) have been cloned from B. napus, representing alleles of two different gene loci, Rlm2-LepR3 and Rlm4-7-9. We report on the development and validation of Kompetitive Allele-Specific PCR (KASP) markers that can discriminate between the resistant and susceptible alleles of each resistance gene. These markers will provide valuable tools for both researchers and industry through the ability to characterize resistance genes without phenotyping.

Comparing the effectiveness of R genes in a 2-year canola–wheat rotation against Leptosphaeria maculans, the causal agent of blackleg disease in Brassica species

Comparing the effectiveness of R genes in a 2-year canola–wheat rotation against Leptosphaeria maculans, the causal agent of blackleg disease in Brassica species

Efficacy of Blackleg Major Resistance Genes in B. napus in Germany.

Leptosphaeria maculans is one of the major pathogens of oilseed rape (B. napus). It causes blackleg disease, which accounts for significant yield losses worldwide. Using cultivars that harbor major resistance (R) genes is one of the most effective control methods. However, the efficacy of major R genes is related to the frequency of the corresponding avirulence (Avr) genes in a L. maculans population. In this paper, we report the Avr profiles of L. maculans populations and the ratio of its mating types in Northern and Central regions of Germany. Eleven Avr genes in five-hundred and seventy-four isolates were characterized either by applying cotyledon tests on a B. napus differential set or by amplifying avirulence gene-specific PCR markers. Fifty-two races were determined, among which the most dominant race was Avrlm6, -7, -11, AvrlepR1, -R2. Results showed that the resistance gene Rlm2 is 100% ineffective, some other major R genes such as Rlm1, Rlm3, Rlm4 and LepR3 are partially effective (with corresponding Avr frequencies ≤ 42%), while LepR1, LepR2, Rlm6, Rlm11 and Rlm7 can still provide relatively effective resistance in the German fields investigated (with corresponding Avr frequencies of 63–100%). Sexual reproduction is a factor that enhances the potential of L. maculans to evolve under selection pressure. Mating types of the L. maculans populations did not deviate from the ratio of 1:1 in the examined regions, indicating that sexual reproduction and ascospores play central roles in the L. maculans lifecycle. Overall, this study provides an important dataset for the establishment of a strategic plan to preserve the efficacies of major R genes in Germany by applying cultivar rotations of oilseed rape.

Novel Brassica hybrids with different resistance to Leptosphaeria maculans reveal unbalanced rDNA signal patterns.

Hybridization of Brassica napus with various Brassicaceae species can result in obtaining new forms with increased resistance to blackleg, a dangerous disease caused mainly by Leptosphaeria maculans. In this study, we aimed to correlate the field resistance of selected Brassica hybrids to blackleg with chromosomal structure revealed by Fluorescence in situ hybridization. Tested genotypes varied in the number of chromosomes and rDNA signals. The greatest variation was observed for A1-type chromosomes. Field evaluation also revealed significant differences in L. maculans resistance. Performed analyses allowed to distinguish three B. napus × Brassica fruticulosa genotypes in which variable patterns of chromosomal structure might be connected to field resistance. However, a more thorough study, including the detection of all A-genome chromosomes, is required.

Independent breakdown events of the Brassica napus Rlm7 resistance gene including via the off-target impact of a dual-specificity avirulence interaction.

Protection of many crops is achieved through the use of genetic resistance. Leptosphaeria maculans, the causal agent of blackleg disease of Brassica napus, has emerged as a model for understanding gene‐for‐gene interactions that occur between plants and pathogens. Whilst many of the characterized avirulence effector genes interact with a single resistance gene in the host, the AvrLm4‐7 avirulence gene is recognized by two resistance genes, Rlm4 and Rlm7. Here, we report the “breakdown” of the Rlm7 resistance gene in Australia, under two different field conditions. The first, and more typical, breakdown probably resulted from widescale use of Rlm7‐containing cultivars whereby selection has led to an increase of individuals in the L. maculans population that have undergone repeat‐induced point (RIP) mutations at the AvrLm4‐7 locus. This has rendered the AvrLm4‐7 gene ineffective and therefore these isolates have become virulent towards both Rlm4 and Rlm7. The second, more atypical, situation was the widescale use of Rlm4 cultivars. Whilst a single‐nucleotide polymorphism is the more common mechanism of virulence towards Rlm4, in this field situation, RIP mutations have been selected leading to the breakdown of resistance for both Rlm4 and Rlm7. This is an example of a resistance gene being rendered ineffective without having grown cultivars with the corresponding resistance gene due to the dual specificity of the avirulence gene. These findings highlight the value of pathogen surveillance in the context of expanded knowledge about potential complexities for Avr–R interactions for the deployment of appropriate resistance gene strategies.

Influence of Elevated Temperatures on Resistance Against Phoma Stem Canker in Oilseed Rape.

Cultivar resistance is an important tool in controlling pathogen-related diseases in agricultural crops. As temperatures increase due to global warming, temperature-resilient disease resistance will play an important role in crop protection. However, the mechanisms behind the temperature-sensitivity of the disease resistance response are poorly understood in crop species and little is known about the effect of elevated temperatures on quantitative disease resistance. Here, we investigated the effect of temperature increase on the quantitative resistance of Brassica napus against Leptosphaeria maculans. Field experiments and controlled environment inoculation assays were done to determine the influence of temperature on R gene-mediated and quantitative resistance against L. maculans; of specific interest was the impact of high summer temperatures on the severity of phoma stem canker. Field experiments were run for three consecutive growing seasons at various sites in England and France using twelve winter oilseed rape breeding lines or cultivars with or without R genes and/or quantitative resistance. Stem inoculation assays were done under controlled environment conditions with four cultivars/breeding lines, using avirulent and virulent L. maculans isolates, to determine if an increase in ambient temperature reduces the efficacy of the resistance. High maximum June temperature was found to be related to phoma stem canker severity. No temperature effect on stem canker severity was found for the cultivar ES Astrid (with only quantitative resistance with no known R genes). However, in the controlled environmental conditions, the cultivar ES Astrid had significantly smaller amounts of necrotic tissue at 20°C than at 25°C. This suggests that, under a sustained temperature of 25°C, the efficacy of quantitative resistance is reduced. Findings from this study show that temperature-resilient quantitative resistance is currently available in some oilseed cultivars and that efficacy of quantitative resistance is maintained at increased temperature but not when these elevated temperatures are sustained for a long period.

Emergence of Fungicide Sensitivity in Leptosphaeria maculans Isolates Collected from the Czech Republic to DMI Fungicides

In the Czech Republic, demethylation inhibitors (DMIs) are used both as fungicides in controlling phoma stem canker and as growth regulators. This heavy use can result in the development of resistant isolates. A total of 45 and 286 Leptosphaeria maculans isolates were tested in vitro, using the mycelial growth and microtiter plate assays, respectively. The objective was to determine the sensitivity of L. maculans isolates collected in the Czech Republic to the fungicides tetraconazole, metconazole, and prochloraz. The mean EC50 values with the mycelial growth plate method were 1.33, 0.78, and 0.40 µg mL−1 for tetraconazole, metconazole, and prochloraz, respectively. The mean EC50 values for the microtiter plate assay were 3.01, 0.44, and 0.19 µg mL−1 for tetraconazole, metconazole, and prochloraz, respectively. All three fungicides also had high variation factors that may be due to inserts in the ERG11 promoter region. In addition, cross sensitivity among the three fungicides was observed. Overall, the high variation factors and the PCR (polymerase chain reaction) results observed in this study could signify the presence of resistant isolates in L. maculans Czech populations, especially in isolates tested for sensitivity to tetraconazole.

Effect of Fusarium blight, Phoma rot, and Sclerotinia blight on rapeseed and mustard plant productivity

The article presents the research data on the effect of Fusarium blight, Phoma rot, and Sclerotinia blight on the plant productivity of winter and spring rapeseed, brown mustard, and white mustard. As a result of studies, we found that Fusarium blight (the pathogens are fungi of the genus Fusarium Link) was the most harmful for the spring rapeseed, brown and white mustard – affection by it significantly decreased plant productivity. At 4 points of affection degree of plants, the harmfulness of the disease reached 71.2, 82.4, and 87.2 %, respectively. On winter rapeseed and mustard, plant productivity was considerably decreased by affection by Phoma rot in the form of a stem cancer (the pathogen is a fungus Leptosphaeria maculans (Desm.) Ces. et. De Not) and the stem form of Sclerotinia blight (the pathogen is a fungus Sclerotinia sclerotiorum (Lib.) De Bary.). The harmfulness of these diseases at 4 points of affection degree of plants reached 56.7 % and 66.7 % on rapeseed, and 56.6 % and 70.7 % on mustard, respectively.

Metabolic Capacity Differentiates Plenodomus lingam from P. biglobosus Subclade 'brassicae', the Causal Agents of Phoma Leaf Spotting and Stem Canker of Oilseed Rape (Brassica napus) in Agricultural Ecosystems.

In contrast to the long-lasting taxonomic classification of Plenodomus lingam and P. biglobosus as one species, formerly termed Leptosphaeria maculans, both species form separate monophyletic groups, comprising sub-classes, differing considerably with epidemiology towards Brassicaceae plants. Considering the great differences between P. lingam and P. biglobosus, we hypothesized their metabolic capacities vary to a great extent. The experiment was done using the FF microplates (Biolog Inc., Hayward, CA, USA) containing 95 carbon sources and tetrazolium dye. The fungi P. lingam and P. biglobosus subclade ‘brassicae’ (3 isolates per group) were cultured on PDA medium for 6 weeks at 20 °C and then fungal spores were used as inoculum of microplates. The test was carried out in triplicate. We have demonstrated that substrate richness, calculated as the number of utilized substrates (measured at λ490 nm), and the number of substrates allowing effective growth of the isolates (λ750 nm), showed significant differences among tested species. The most efficient isolate of P. lingam utilized 36 carbon sources, whereas P. biglobosus utilized 60 substrates. Among them, 25–29 carbon sources for P. lingam and 34–48 substrates for P. biglobosus were efficiently used, allowing their growth. Cluster analysis based on Senath criteria divided P. biglobosus into two groups and P. lingam isolates formed one group (33% similarity). We deduce the similarities between the tested species help them coexist on the same host plant and the differences greatly contribute to their different lifestyles, with P. biglobosus being less specialized and P. lingam coevolving more strictly with the host plant.