Phoma Medicaginis Research Articles

Unveiling novel contrasting photosynthetic responses in Medicago truncatula under combined drought stress and Phoma medicaginis infection

Forage legumes face simultaneous abiotic and biotic stresses, causing substantial yield losses. This study explores the combined impacts of drought and Phoma medicaginis infection on the growth and photosynthetic activity of Medicago truncatula contrasting lines TN6.18 and F83005.5. The tolerant TN6.18 line exhibits superior tolerance to combined drought and P. medicaginis infection, manifesting in minimal leaf area reduction, the highest number of healthy leaves, increased carotenoids content, a consistently high and stable photosynthesis rate, and enhanced performance of photosystems PSI and PSII. On the contrary, the sensitive F83005.5 line shows pronounced leaf chlorosis, particularly under drought stress, and decreased pigment levels under the combination of drought and P. medicaginis infection stresses (combined stress). Moreover, the drought-stressed F83005.5, experiences reduced hydration and photosynthetic performance, linked to diminished gas exchange and chlorophyll fluorescence parameters. Chlorophyll fluorescence revealed more severe PSI impairment than PSII under combined stress. In conclusion, understanding the Pm8 infection-drought interaction enhances insights into M. truncatula resistance mechanisms to the combination of these stresses.

Response Surface Methodology-Based Optimization of the Chitinolytic Activity of Burkholderia contaminans Strain 614 Exerting Biological Control against Phytopathogenic Fungi.

As part of the development of alternative and environmentally friendly control against phytopathogenic fungi, Burkholderia cepacia could be a useful species notably via the generation of hydrolytic enzymes like chitinases, which can act as a biological control agent. Here, a Burkholderia contaminans S614 strain exhibiting chitinase activity was isolated from a soil in southern Tunisia. Then, response surface methodology (RSM) with a central composite design (CCD) was used to assess the impact of five factors (colloidal chitin, magnesium sulfate, dipotassium phosphate, yeast extract, and ammonium sulfate) on chitinase activity. B. contaminans strain 614 growing in the optimized medium showed up to a 3-fold higher chitinase activity. This enzyme was identified as beta-N-acetylhexosaminidase (90.1 kDa) based on its peptide sequences, which showed high similarity to those of Burkholderia lata strain 383. Furthermore, this chitinase significantly inhibited the growth of two phytopathogenic fungi: Botrytis cinerea M5 and Phoma medicaginis Ph8. Interestingly, a crude enzyme from strain S614 was effective in reducing P. medicaginis damage on detached leaves of Medicago truncatula. Overall, our data provide strong arguments for the agricultural and biotechnological potential of strain S614 in the context of developing biocontrol approaches.

Alfalfa Spring Black Stem and Leaf Spot Disease Caused by Phoma medicaginis: Epidemic Occurrence and Impacts.

Alfalfa spring black stem and leaf spot disease (ASBS) is a cosmopolitan soil-borne and seed-borne disease caused by Phoma medicaginis, which adversely affects the yield, and nutritive value and can stimulate production of phyto-oestrogenic compounds at levels that may adversely affect ovulation rates in animals. This review summarizes the host range, damage, and symptoms of this disease, and general features of the infection cycle, epidemic occurrence, and disease management. ASBS has been reported from over 40 countries, and often causes severe yield loss. Under greenhouse conditions, reported yield loss was 31-82% for roots, 32-80% for leaves, 21% for stems and 26-28% for seedlings. In field conditions, the forage yield loss is up to 56%, indicating that a single-cut yield of 5302 kg/ha would be reduced to 2347 kg/ha. P. medicaginis can infect up to 50 species of plants, including the genera Medicago, Trifolium, Melilotus, and Vicia. ASBS is more severe during warm spring conditions before the first harvest than in hot summer and cooler winter conditions, and can infect alfalfa roots, stems, leaves, flowers, pods, and seeds, with leaf spot and/or black stem being the most typical symptoms. The primary infection is caused by the overwintering spores and mycelia in the soil, and on seeds and the cortex of dead and dry stems. The use of resistant cultivars is the most economical and effective strategy for the control of ASBS. Although biological control has been studied in the glasshouse and is promising, chemical control is the main control method in agriculture.

Discovering new genes for alfalfa (Medicago sativa) growth and biomass resilience in combined salinity and Phoma medicaginis infection through GWAS.

Salinity and Phoma medicaginis infection represent significant challenges for alfalfa cultivation in South Africa, Europe, Australia, and, particularly, Tunisia. These constraints have a severe impact on both yield and quality. The primary aim of this study was to establish the genetic basis of traits associated with biomass and growth of 129 Medicago sativa genotypes through genome-wide association studies (GWAS) under combined salt and P. medicaginis infection stresses. The results of the analysis of variance (ANOVA) indicated that the variation in these traits could be primarily attributed to genotype effects. Among the test genotypes, the length of the main stem, the number of ramifications, the number of chlorotic leaves, and the aerial fresh weight exhibited the most significant variation. The broad-sense heritability (H²) was relatively high for most of the assessed traits, primarily due to genetic factors. Cluster analysis, applied to morpho-physiological traits under the combined stresses, revealed three major groups of accessions. Subsequently, a GWAS analysis was conducted to validate significant associations between 54,866 SNP-filtered single-nucleotide polymorphisms (SNPs) and seven traits. The study identified 27 SNPs that were significantly associated with the following traits: number of healthy leaves (two SNPs), number of chlorotic leaves (five SNPs), number of infected necrotic leaves (three SNPs), aerial fresh weight (six SNPs), aerial dry weight (nine SNPs), number of ramifications (one SNP), and length of the main stem (one SNP). Some of these markers are related to the ionic transporters, cell membrane rigidity (related to salinity tolerance), and the NBS_LRR gene family (associated with disease resistance). These findings underscore the potential for selecting alfalfa genotypes with tolerance to the combined constraints of salinity and P. medicaginis infection.

Plant growth stage and Phoma medicaginis inoculum concentration together determine severity of Phoma black stem and leaf spot and consequent phytoestrogen production in annual Medicago spp.

AbstractPhoma black stem and leaf spot disease (Phoma medicaginis) not only destroys annual Medicago spp. forage and seed yield but also reduces herbage quality by consequent phytoestrogen production that reduces ovulation of grazing animals. Two controlled environment studies evaluated the effects of plant developmental stage in annual Medicago rugosa ‘Paraponto’ and M. scutellata ‘Sava’ and different inoculum concentrations of P. medicaginis in M. littoralis ‘Harbinger’ and M. polymorpha ‘Serena’ on disease development and coumestrol production. Disease incidence and severity and coumestrol production were dependent on plant developmental stage, cultivar and inoculum level (all p ≤ 0.001). Disease was least in 4‐week‐old plants; highest coumestrol was in inoculated 10‐week‐old Sava (1353 mg/kg) and least coumestrol in uninoculated 4‐week‐old Paraponto (87 mg/kg); and there was a positive correlation of disease incidence/severity factors with coumestrol across cultivars and plant growth stages (p < 0.001). Disease levels and coumestrol production were determined by inoculum concentration and cultivar (both p ≤ 0.001). Highest coumestrol was in Serena inoculated with 107 conidia/mL (265 mg/kg); lowest coumestrol was in uninoculated Harbinger (6 mg/kg); and there was a significant positive correlation of disease incidence/severity factors with coumestrol across cultivars and inoculum concentrations (p < 0.001). These studies emphasize both the opportunity for farmers to better use annual Medicago spp. stands for grazing reproducing animals early in the growing season when both disease and coumestrol levels are lowest, and the need for heightened farmer vigilance at later growth stages with greater disease and consequent phytoestrogen risk for grazing animals.

Arbuscular mycorrhizal fungus changes alfalfa (Medicago sativa) metabolites in response to leaf spot (Phoma medicaginis) infection, with subsequent effects on pea aphid (Acyrthosiphon pisum) behavior.

Plant disease occurs simultaneously with insect attack. Arbuscular mycorrhizal fungi (AMF) modify plant biotic stress response. Arbuscular mycorrhizal fungi and pathogens may modify plant volatile organic compound (VOC) production and insect behavior. Nevertheless, such effects are rarely studied, particularly for mesocosms where component organisms interact with each other. Plant-mediated effects of leaf pathogen (Phoma medicaginis) infection on aphid (Acyrthosiphon pisum) infestation, and role of AMF (Rhizophagus intraradices) in modifying these interactions were elucidated in a glasshouse experiment. We evaluated alfalfa disease occurrence, photosynthesis, phytohormones, trypsin inhibitor (TI) and total phenol response to pathogen and aphid attack, with or without AMF, and aphid behavior towards VOCs from AMF inoculated and non-mycorrhizal alfalfa, with or without pathogen infection. AM fungus enhanced alfalfa resistance to pathogen and aphid infestation. Plant biomass, root : shoot ratio, net photosynthetic rate, transpiration rate, stomatal conductance, salicylic acid, and TI were significantly increased in AM-inoculated alfalfa. Arbuscular mycorrhizal fungi and pathogen significantly changed alfalfa VOCs. Aphids preferred VOCs of AM-inoculated and nonpathogen-infected to nonmycorrhizal and pathogen-infected alfalfa. We propose that AMF alter plant response to multiple biotic stresses in ways both beneficial and harmful to the plant host, providing a basis for strategies to manage pathogens and herbivore pests.

Variability in Responses to Phoma medicaginis Infection in a Tunisian Collection of Three Annual Medicago Species

Spring black stem and leaf spot, caused by Phoma medicaginis, is an issue in annual Medicago species. Therefore, in this study, we analyzed the response to P. medicaginis infection in a collection of 46 lines of three annual Medicago species (M. truncatula, M. ciliaris, and M. polymorpha) showing different geographic distribution in Tunisia. The reaction in the host to the disease is explained by the effects based on plant species, lines nested within species, treatment, the interaction of species × treatment, and the interaction of lines nested within species × treatment. Medicago ciliaris was the least affected for aerial growth under infection. Furthermore, the largest variation within species was found for M. truncatula under both conditions. Principal component analysis and hierarchical classification showed that M. ciliaris lines formed a separate group under control treatment and P. medicaginis infection and they are the most vigorous in growth. These results indicate that M. ciliaris is the least susceptible in response to P. medicaginis infection among the three Medicago species investigated here, which can be used as a good candidate in crop rotation to reduce disease pressure in the field and as a source of P. medicaginis resistance for the improvement of forage legumes.

Surfactin and Spo0A-Dependent Antagonism by Bacillus subtilis Strain UD1022 against Medicago sativa Phytopathogens.

Plant growth-promoting rhizobacteria (PGPR) such as the root colonizers Bacillus spp. may be ideal alternatives to chemical crop treatments. This work sought to extend the application of the broadly active PGPR UD1022 to Medicago sativa (alfalfa). Alfalfa is susceptible to many phytopathogens resulting in losses of crop yield and nutrient value. UD1022 was cocultured with four alfalfa pathogen strains to test antagonism. We found UD1022 to be directly antagonistic toward Collectotrichum trifolii, Ascochyta medicaginicola (formerly Phoma medicaginis), and Phytophthora medicaginis, and not toward Fusarium oxysporum f. sp. medicaginis. Using mutant UD1022 strains lacking genes in the nonribosomal peptide (NRP) and biofilm pathways, we tested antagonism against A. medicaginicola StC 306-5 and P. medicaginis A2A1. The NRP surfactin may have a role in the antagonism toward the ascomycete StC 306-5. Antagonism toward A2A1 may be influenced by B. subtilis biofilm pathway components. The B. subtilis central regulator of both surfactin and biofilm pathways Spo0A was required for the antagonism of both phytopathogens. The results of this study indicate that the PGPR UD1022 would be a good candidate for further investigations into its antagonistic activities against C. trifolii, A. medicaginicola, and P. medicaginis in plant and field studies.

Arbuscular mycorrhizal fungus changes alfalfa response to pathogen infection activated by pea aphid infestation.

Arbuscular mycorrhizal (AM) fungi are important for the resistance of plants to insect infestation and diseases. However, the effect of AM fungal colonization of plants response to pathogen infection activated by pea aphid infestation is unknown. Pea aphid (Acyrthosiphon pisum) and the fungal pathogen Phoma medicaginis severely limit alfalfa production worldwide. This study established an alfalfa (Medicago sativa)-AM fungus (Rhizophagus intraradices)-pea aphid-P. medicaginis experimental system to clarify the effects of an AM fungus on the host plant response to insect infestation and subsequent fungal pathogen infection. Pea aphid increased the disease incidence of P. medicaginis by 24.94%. The AM fungus decreased the disease index by 22.37% and enhanced alfalfa growth by increasing the uptake of total nitrogen and total phosphorus. The aphid induced polyphenol oxidase activity of alfalfa, and the AM fungus enhanced plant-defense enzyme activity against aphid infestation and subsequent P. medicaginis infection. In addition, the AM fungus increased the contents of jasmonic acid and abscisic acid in plants exposed to aphid infestation or pathogen infection. Abscisic acid and genes associated with the gene ontology term "hormone binding" were upregulated in aphid-infested or pathogen-infected alfalfa. The results demonstrate that an AM fungus enhances plant defense and signaling components induced by aphid infestation, which may contribute to improved defense against subsequent pathogen infection.

Temperature and Soil Moisture Drive Coumestrol Concentration in Annual Medicago spp. in the Presence but Not Absence of Phoma Black Stem and Leaf Spot (Phoma medicaginis)

Studies were undertaken to determine the impact of environmental variables temperature (12.5/9.5, 20/17, 27/24 °C day/night) and soil moisture (100, 50% WHC), and their interaction with Phoma medicaginis infection, on production of the phytoestrogen coumestrol in annual Medicago rugosa cv. Paraponto and M. scutellata cv. Sava. Disease factors measured included leaf disease incidence/severity, petiole/stem disease incidence/severity, and leaf yellowing severity. Coumestrol levels were determined using gas chromatography–mass spectrometry (GC–MS). Increasing temperature from 12.5/9.5 °C to 27/24 °C in inoculated plants significantly (p < 0.05) increased coumestrol from 193 mg kg−1 to 390 mg kg−1, but there were no differences in coumestrol production across all three temperatures in uninoculated plants. Reducing soil moisture from 100% to 50% WHC at the highest temperature (27/24 °C) caused the greatest increase in coumestrol production from 156 to 269 mg kg−1 in inoculated plants. The greatest coumestrol production (600 mg kg−1) was under 27/24 °C/50% WHC for Sava infected with P. medicaginis and least coumestrol (1.6 mg kg−1) was Sava under 20/17 °C/50% WHC in the absence of P. medicaginis. Clearly, situations of higher temperatures in conjunction with lower soil moisture levels cause greatest elevation in coumestrol in the presence of P. medicaginis, levels far exceeding the animal risk threshold of 25 mg kg−1.

Phoma black stem severity and phytoestrogen production in annual Medicago spp. is primarily determined by interaction of cultivar and pathogen isolate

AbstractPhoma black stem and leaf spot disease (caused by Phoma medicaginis) not only diminishes forage and seed yield but stimulates production of detrimental phytoestrogens in annual Medicago spp. This study aimed to evaluate relationships between disease development from five isolates of P. medicaginis on 16 cultivars with production of coumestrol and 4′‐O‐methylcoumestrol. In the presence of P. medicaginis, Sava had the highest coumestrol and 4′‐O‐methylcoumestrol (640 and 85 mg/kg, respectively) followed by Caliph (253 and 15 mg/kg, respectively). In the absence of P. medicaginis, Jemalong and Paragosa showed highest and lowest coumestrol (137 and 0 mg/kg, respectively). 4′‐O‐methylcoumestrol was not produced in disease‐free plants, but coumestrol was. Disease incidence and severity on leaves and on petiole/stems, and consequent leaf yellowing severity ranged from 5%–98.7%, 0%–100%, 4.4%–98.7%, 1.7%–100%, and 0%–85%. Sava, Paraponto, Harbinger, and Serena were most susceptible, while Tornafield and Caliph were least susceptible. There was significant overall positive correlation of disease incidence/severity factors across cultivars (p < 0.01) with both coumestrol and 4′‐O‐methylcoumestrol. Jemalong was least responsive and Paragosa and Sava most responsive to coumestrol production following P. medicaginis inoculation. Coumestrol in inoculated Paragosa increased to 373 mg/kg in comparison with 0 mg/kg in controls. These findings are of critical importance towards developing less disease‐susceptible annual Medicago spp. producing less detrimental phytoestrogens. Least susceptible cultivars like Tornafield and Caliph can be used to manage yield loss, whilst least responsive cultivars to phytoestrogen production like Caliph also can help to reduce phytoestrogen production.

Sequential infections by 32 isolates of

Context Studies of Phoma black stem and leaf spot disease (caused by Phoma medicaginis) in annual medics (Medicago spp.) normally involve a ‘once-only’ inoculation not reflecting multiple pathogen infection and phytoestrogen production cycles in the field. Phytoestrogen production by plants can result in lower ovulation rates in grazing animals. Aims We aimed to determine whether sequential infections by P. medicaginis increase production of phytoestrogens in annual medics, and to measure the genetic diversity of isolates. Methods In a greenhouse experiment, pathogenicity and virulence were investigated across 32 isolates of P. medicaginis following one, two or three rounds of inoculation of M. polymorpha var. brevispina. Production of the phytoestrogens coumestrol and 4′-O-methyl coumestrol was measured, and correlation with disease parameters assessed. DNA sequencing using ITS, β-tubulin, calmodulin and P. medicaginis-specific EFNI-1α was applied for phylogenetic analysis of isolates from Western Australia and elsewhere. Key results Across isolates, highest leaf disease incidence was 76%, petiole disease incidence 61%, leaf disease severity 52% and petiole disease severity 53%. Stem coumestrol content range was 45–1247 mg kg−1, and 4′-O-methyl coumestrol 0–344 mg kg−1. All measures were highest after three rounds of inoculation. Overall, there was a positive correlation of leaf disease incidence with coumestrol content (P < 0.05) and of both leaf and petiole disease incidence with 4′-O-methylcoumestrol content (P < 0.01, P < 0.05, respectively). Phylogenetic analysis revealed a high degree of genetic similarity among Western Australian isolates, generally grouping into a single separate cluster across the four markers, and genetically distinct from isolates sourced outside Australia. Conclusions Leaf disease incidence was the best discriminating disease parameter for coumestrol and 4′-O-methylcoumestrol content. Western Australian isolates of P. medicaginis were genetically similar and unique, possibly due to geographic separation. Implications The study emphasised the importance of sequential inoculations when screening annual Medicago genotypes towards developing cultivars with superior disease resistance and enhanced animal reproductive outcomes.

Phoma medicaginis Isolate Differences Determine Disease Severity and Phytoestrogen Production in Annual Medicago spp.

Phoma black stem and leaf spot disease of annual Medicago spp., caused by Phoma medicaginis, not only can devastate forage and seed yield but can reduce herbage quality by inducing production of phytoestrogens (particularly coumestrol and 4'-O-methylcoumestrol), which can also reduce the ovulation rates of animals grazing infected forage. We determined the consequent phytoestrogen levels on three different annual Medicago species/cultivars (Medicago truncatula cultivar Cyprus, Medicago polymorpha var. brevispina cultivar Serena, and Medicago murex cultivar Zodiac) after inoculation with 35 isolates of P. medicaginis. Across the isolate × cultivar combinations, leaf disease incidence, petiole/stem disease incidence, leaf disease severity, petiole disease severity, and leaf yellowing severity ranged up to 100, 89.4, 100, 58.1, and 61.2%, respectively. Cultivars Cyprus and Serena were the most susceptible and cultivar Zodiac was the most resistant to P. medicaginis. Isolates WAC3653, WAC3658, and WAC4252 produced the most severe disease. Levels of phytoestrogens in stems ranged from 25 to 1,995 mg/kg for coumestrol and from 0 to 418 mg/kg for 4'-O-methylcoumestrol. There was a significant positive relationship of disease incidence and severity parameters with both coumestrol and 4'-O-methylcoumestrol contents, as noted across individual cultivars and across the three cultivars overall, where r = 0.39 and 0.37 for coumestrol and 4'-O-methylcoumestrol, respectively (P < 0.05). Although cultivar Serena was most susceptible to P. medicaginis and produced the highest levels of phytoestrogens in the presence of P. medicaginis, cultivar Zodiac contained the highest levels of phytoestrogens in comparison with other cultivars in the absence of P. medicaginis. There was a 15-fold increase in coumestrol in cultivar Serena but only a 7-fold increase in cultivar Zodiac from infection of P. medicaginis. The study highlights that the intrinsic ability of a particular cultivar to produce phytoestrogens in the absence of the pathogen, and its comparative ability to produce phytoestrogens in the presence of the P. medicaginis, are both important and highly relevant to developing new annual Medicago spp. cultivars that offer improved disease resistance and better animal reproductive outcomes.

Variation of Medicago sativa varieties tolerance to Phoma medicaginis infection.

Due to its very important agronomic value and nutritional quality, Medicago sativa L. is considered as the queen of fodder and the first cultivated forage crop in the world. In field conditions, M. sativa is exposed to several biotic and/or abiotic constraints that affect its quality. In this regard, research is still underway to improve M. sativa resistance to many biotic stresses and, in this context, we analyzed the responses of a core collection of 10 varieties of M. sativa to Phoma medicaginis infection. Results from ANOVA showed that most growth parameters exhibited significant differences between the studied varieties. Nevertheless, only the number of healthy leaves among infection parameters varied significantly between the varieties. The local variety Gabès2355 exhibited the highest biomass. Positive correlations were found between the measured parameters. PCA based on the traits showing significant differences among the studied lines showed that the Gabès variety formed a separate group. Cluster analysis revealed that the studied varieties are classified into three major groups. The first group is formed by Gabès2353, the second group is composed of the Californian and El Hamma varieties, and the third group is constituted of the seven remaining varieties. Gabès2355 was the most tolerant to the Pm8 strain of P. medicaginis while Magna601 variety was the most susceptible. These two varieties will be useful to analyze the physiological and genetic determinants for M. sativa tolerance to P. medicaginis infection.

Plant quarantine measures for the safe global distribution of germplasm of ICRISAT mandate crops

ICRISAT safely exported 182,605 seed samples to 127 countries during 1998–2019. The maximum number of exported samples were of sorghum (51,309) followed by chickpea (48,513) and pearl millet (39,771). A total of 2749 seed samples (chickpea 1301, groundnut 300, minor millets 50, pearl millet 338, pigeonpea 395 and sorghum 365) were rejected due to presence of seed-borne pathogens of quarantine significance. Thirty-one pathogenic fungal species were detected in the blotter test and seven pathogens detected in seed washing and sedimentation test. Maximum fungal/oomycetes species were detected in sorghum seed samples (18) followed by chickpea (16), pigeonpea (15), groundnut (10), pearl millet (9) and minor millets (6). Acremonium sp., Botrytis sp., Cercospora sp., Colletotrichum sp., Exserohilum turcicum, Fusarium sp., Phoma medicaginis, Phomopsis sp., Pyricularia grisea, Sclerotium rolfsii, Peronosclerospora sp., Sporisorium sorghi, Puccinia purpurea, Moesziomyces penicillariae, Claviceps fusiformis and Sphacelotheca sp. were detected in the seed samples.

High diversity of responses among Medicago truncatula lines to Phoma medicaginis infection.

Medicago truncatula is an omni-Mediterranean species grown as an annual forage legume. In addition to its small genome size and simple genetics, M. truncatula harbors several attributes, which make it an attractive model forage plant. In this study, we investigated the variation of responses in ten parental lines of M. truncatula to Phoma medicaginis infection. Plants were cultivated in the growth chamber under controlled conditions and were inoculated after two months with P. medicaginis. At harvest, 13 quantitative traits of growth and pathogenicity were measured. Results from ANOVA showed that the variation of analyzed parameters was explained by the effect of line. All measured parameters, except the root fresh weight, showed significant difference among the 10 studied lines. Most tolerant lines are those with the lowest ratios of the number of infected and dead leaves. Studied lines were clustered into three groups according to their responses to P. medicaginis infection. The most resistant TN6.18 line and most sensitive F83005.5 to P. medicaginis are useful for the exploration of physiological mechanisms and genetic determinants for M. truncatula tolerance to this constraint.

Competition and facilitation among fungal plant parasites affect their life‐history traits

Multi‐infections may result in either competitive exclusion or coexistence on the same host of pathogen genotypes belonging to the same or different species. Epidemiological consequences of multiple infections, particularly how the development and transmission of a pathogen can be modified by the presence of another pathogen, are well documented. However, understanding how life history strategies of each pathogen modulate co‐infection outcomes remains quite elusive. To analyze how co‐infection drives changes in life history traits and affects co‐existence in epidemic pathogens, we infected detached pea stipules with two fungal species, Peyronellaea pinodes and Phoma medicaginis var. pinodella (considering two strains per species), part of the ascochyta blight complex but presenting different life history strategies. All pairwise combinations (including self‐pairs) between two strains of each species were tested. Strains were inoculated simultaneously, but apart from one another on the stipule. For each strain, four life history traits were measured: incubation period, necrosis area six days after inoculation, latent period and offspring production. Results show that, in co‐infection, when resources are highly allocated to lesion development, the time between inoculation and the appearance of reproduction structures (latent period) and offspring production decreased, and vice‐versa relative to single infections. The direction and/or magnitude of these responses to co‐infection depend on the co‐infecting strains. Moreover, these changes were always higher in self‐pairs than in mixed co‐infections. These results suggest facilitation between co‐infecting strains, resulting in the selection of an intermediate level of virulence (here measured as the lesion development) at the expense of pathogen offspring production. This strategy allows the development and reproduction of each co‐infecting strain when sharing limited resources. However, the direction and strength of these life history traits variations in co‐infection depend on the life history strategy of the co‐infecting strains, with a clear difference between ‘opportunists', ‘scavengers' and ‘pioneer colonisers'.

Identification of fungal pathogens and analysis of genetic diversity of Fusarium tricinctum causing root rots of alfalfa in north‐east China

AbstractAlfalfa root rot is a devastating disease complex found worldwide. Population structure and genetic diversity of fungal pathogens causing alfalfa root rot in north‐east China are not well understood. In this study, 480 fungal isolates were collected from six major alfalfa‐growing regions in Heilongjiang province, China. They were identified as Fusarium tricinctum, F. oxysporum, F. acuminatum, F. solani, F. equiseti, Phoma medicaginis, Plectosphaerella cucumerina, Alternaria alternata, and Chaetomium globosum and caused root rot on alfalfa in greenhouse studies. F. tricinctum was the predominant species among the isolates, and P. medicaginis and C. globosum had not previously been reported causing alfalfa root rot in north‐east China. Of the 73 F. tricinctum isolates identified, the majority were moderately or highly aggressive on alfalfa. No isolate of F. tricinctum was sensitive to carbendazim (1 and 10 μg/ml), indicating that, although commonly used, it is not suitable for management of the disease in this area. F. tricinctum isolates were analysed using AFLP markers and divided into eight genetic groups with 28 pairs of primers. Analysis of molecular variance indicated significant correlation between genetic groups of F. tricinctum isolates and their geographical locations or aggressiveness. Pairwise comparison and STRUCTURE analysis also indicated that geographical locations and aggressiveness of isolates had a significant effect on population differentiation. This study provides insight into the genetic diversity and reproductive biology of F. tricinctum, enhances understanding of the population diversity of alfalfa root rot pathogens in north‐east China, and facilitates development of effective strategies for managing this destructive disease complex.

Early detection of Ascochyta blight (Ascochyta rabiei) of chickpea by traditional PCR

Ascochyta blight is the major disease affecting chickpea (Cicer arietinum) around the world. Since the first report of Ascochyta rabiei's isolation in Argentina in 2012, the pathogen has caused severe economic losses in crop production; so, the detection and rapid identification of the pathogen in early stages is key for the management of the disease. In this work, a traditional PCR procedure for detection of A. rabiei directly from plant tissues has been described based on beta-tubulin gene. The TP-6/TP-9 specific primers designed, amplified only a single PCR band of 770 bp from A. rabiei. The specificity of the primers was checked using 12 isolates of A. rabiei and DNA from 10 other different fungi including common pathogens of chickpea as Alternaria alternata, Botrytis cinerea, Sclerotinia sclerotiorum and Phoma medicaginis that cause similar symptoms. The detection sensitivity with primers was 2 × 104 ng μl−1 genomic DNA. In inoculated plant material, PCR amplification gave a band of the expected size and no amplification was observed when DNA was from healthy and uninoculated plants. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods. The PCR-based method developed here can simplify both plant disease diagnosis, and pathogen monitoring in an early phase, as well as aid in effective management practices that avoid the disease advance and minimize losses.

Functional analysis of Medicago-derived pathogen-induced gene promoters for usage in transgenic alfalfa

Three gene promoters from the model legume species Medicago truncatula and Medicago sativa (alfalfa) pathogenesis-related (PR) proteins (MtPR5, MtPR10, and MsPR10) were isolated and investigated using in silico and in situ approaches. For the functional analysis of these promoters, plant transformation vectors linking promoter sequences with the β-glucuronidase (GUS) reporter gene were constructed and utilized to generate transgenic alfalfa. Histochemical GUS staining established that the PR5 and both PR10 promoters were functional in alfalfa and induce GUS expression primarily in the root tissue. When transgenic alfalfa leaves were inoculated with a diverse set of alfalfa pathogens, either Phoma medicaginis, Colletotrichum trifolii, or Pseudomonas syringae pv. syringae, the promoters were responsive to induction by both bacterial and fungal pathogens. Gene expression analysis also indicated that all three PR promoters were pathogen-induced and upregulate transgene expression. Additionally, several putative transcription regulator elements (REs) responsible for the observed pathogen-induced promoter activity were predicted in each of the promoter sequences. These characterized Medicago PR promoters may be a unique and valuable tool for pathogen-induced transgenic expression of antimicrobial proteins or other genes enhancing disease resistance in alfalfa.