Phytophthora Medicaginis Research Articles

Using RNA sequencing to unravel molecular changes underlying the defense response in chickpea induced by Phytophthora medicaginis.

Phytophthora root rot (PRR), caused by Phytophthora medicaginis, is a major soil-borne disease of chickpea in Australia. Breeding for PRR resistance is an effective approach to avoid significant yield loss. Genetic resistance has been identified in cultivated chickpea (Cicer arietinum) and in the wild relative C. echinospermum, with previous studies identifying independent genetic loci associated with each of these sources. However, the molecular mechanisms associated with PRR resistance are not known. RNA sequencing analysis employed in this study identified changes in gene expression in roots of three chickpea genotypes grown hydroponically, early post-infection with P. medicaginis zoospores. Analyses of differentially expressed genes (DEG) identified the activation of a higher number of non-specific R-genes in a PRR-susceptible variety than in the resistant genotypes, suggesting a whole plant resistance response occurring in chickpea against the pathogen. Contrasting molecular changes in signaling profiles, proteolysis and transcription factor pathways were observed in the cultivated and wild Cicer-derived resistant genotypes. DEG patterns supported a hypothesis that increased root elongation and reduced adventitious root formation limit the pathogen entry points in the genotype containing the wild Cicer source of PRR resistance. Candidate resistance genes, including an aquaporin and a maltose transporter in the wild Cicer source and GDSL esterases/lipases in the cultivated source of resistance, were oppositely regulated. Increased knowledge of these genes and pathways will improve our understanding of molecular mechanisms controlling PRR resistance in chickpea, and support the development of elite chickpea varieties through molecular breeding approaches.

Selection for Phytophthora Root Rot Resistance in Chickpea Crosses Affects Yield Potential of Chickpea × Cicer echinospermum Backcross Derivatives.

Phytophthora root rot (PRR) of chickpea (Cicer arietinum) caused by Phytophthora medicaginis is an important disease. Partial resistance to PRR is sourced from Cicer echinospermum. In this study, we evaluated if lines with low levels of PRR foliage symptoms in two contrasting recombinant inbred line (RIL) populations parented by chickpea cultivars (Yorker and Rupali) and 04067-81-2-1-1 (C. echinospermum, interspecific breeding line) had a significant drag on yield parameters. For the Yorker × 04067-81-2-1-1 population with the highest level of PRR resistance, in the absence of PRR, low foliage symptom RIL had significantly later flowering and podding, lower grain yields, and lighter seed and shorter plant phenotypes than high foliage symptom RIL. A quantitative trait locus analysis identified significant QTL for flowering, height, 100-seed weight, and yield, and there was a significantly higher frequency of alleles for the negative agronomic traits (i.e., drag) from the 04067-81-2-1-1 parent in low foliage symptom RIL than in high foliage symptom RIL. For the Rupali × 04067-81-2-1-1 population with lower levels of PRR resistance, in the absence of PRR, low foliage symptom RIL had significantly lighter seed and shorter plants than high foliage symptom RIL. Significant QTL were detected, the majority were for the timing of flowering and podding (n = 18), others were for plant height, yield, and 100-seed weight. For this second population, the frequency of alleles for the negative agronomic traits from the 04067-81-2-1-1 parent did not differ between low and high foliage symptom RIL. The 100 seed weight of RIL under moderate PRR disease pressure showed some promise as a yield component trait to identify phenotypes with both high levels of PRR resistance and grain yield potential for further seed number evaluations. We identified that large population sizes are required to enable selection among chickpea × C. echinospermum crosses for high levels of PRR resistance without a significant drag on yield.

Functional genomics identifies a small secreted protein that plays a role during the biotrophic to necrotrophic shift in the root rot pathogen Phytophthora medicaginis.

Hemibiotrophic Phytophthora are a group of agriculturally and ecologically important pathogenic oomycetes causing severe decline in plant growth and fitness. The lifestyle of these pathogens consists of an initial biotrophic phase followed by a switch to a necrotrophic phase in the latter stages of infection. Between these two phases is the biotrophic to necrotrophic switch (BNS) phase, the timing and controls of which are not well understood particularly in Phytophthora spp. where host resistance has a purely quantitative genetic basis. To investigate this we sequenced and annotated the genome of Phytophthora medicaginis, causal agent of root rot and substantial yield losses to Fabaceae hosts. We analyzed the transcriptome of P. medicaginis across three phases of colonization of a susceptible chickpea host (Cicer arietinum)and performed co-regulatory analysis to identify putative small secretedprotein (SSP) effectors that influence timing of the BNS in a quantitative pathosystem. The genome of P. medicaginis is ~78 Mb, comparable to P. fragariae and P. rubi which also cause root rot. Despite this, it encodes the second smallest number of RxLR (arginine-any amino acid-leucine-arginine) containing proteins of currently sequenced Phytophthora species. Only quantitative resistance is known in chickpea to P. medicaginis, however, we found that many RxLR, Crinkler (CRN), and Nep1-like protein (NLP) proteins and carbohydrate active enzymes (CAZymes) were regulated during infection. Characterization of one of these, Phytmed_10271, which encodes an RxLR effector demonstrates that it plays a role in the timing of the BNS phase and root cell death. These findings provide an important framework and resource for understanding the role of pathogenicity factors in purely quantitative Phytophthora pathosystems and their implications to the timing of the BNS phase.

Rhizosphere and Root Community Analysis of Oomycetes Associated with Poor Alfalfa (Medicago sativa) Seedling Establishment

Alfalfa ( Medicago sativa) seeds planted in cold, wet soil are prone to seed rot, pre- and postemergence damping-off, and seedling root rot. These diseases reduce initial stand density and affect forage yields and winter survival when root systems are stunted by infection. Successful management depends on identifying the causal agents but isolation in pure culture is time consuming and may fail to recover slow-growing organisms or those requiring special growth conditions. Here, the oomycetes in rhizosphere soil and roots from eight locations with a history of poor alfalfa establishment were identified by amplicon sequence analysis. Seedling bioassays were done with bulk soil and quantitative PCR (qPCR) assays were done using DNA from bulk soil, rhizosphere soil, and the root tissue for Aphanomyces euteiches, Phytophthora medicaginis, Pythium irregulare, P. sylvaticum, and P. ultimum var. ultimum. In the root tissue and rhizosphere, A. euteiches was the dominant taxon, followed by Phytophthora medicaginis and P. sansomeana, a broad-host-range pathogen not previously known to infect alfalfa. Pythium inflatum and Globisporangium perplexum were also significantly overrepresented in roots at five of the eight locations, indicating that these taxa are commonly enriched in alfalfa roots but had not previously been identified as alfalfa pathogens. The qPCR assays of bulk soil showed that A. euteiches and Phytophthora medicaginis were unevenly distributed in the plots and low concentrations of the pathogens led to high levels of root rot disease. These results support development of cultivars with high levels of resistance to the most common oomycete pathogens.

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.

Inoculum production of Phytophthora medicaginis can be used to screen for partial resistance in chickpea genotypes.

Phytophthora root rot caused by Phytophthora medicaginis is an important disease of chickpeas (Cicer arietinum) in Australia with limited management options, increasing reliance on breeding for improved levels of genetic resistance. Resistance based on chickpea-Cicer echinospermum crosses is partial with a quantitative genetic basis provided by C. echinospermum and some disease tolerance traits originating from C. arietinum germplasm. Partial resistance is hypothesised to reduce pathogen proliferation, while tolerant germplasm may contribute some fitness traits, such as an ability to maintain yield despite pathogen proliferation. To test these hypotheses, we used P. medicaginis DNA concentrations in the soil as a parameter for pathogen proliferation and disease assessments on lines of two recombinant inbred populations of chickpea-C. echinospermum crosses to compare the reactions of selected recombinant inbred lines and parents. Our results showed reduced inoculum production in a C. echinospermum backcross parent relative to the C. arietinum variety Yorker. Recombinant inbred lines with consistently low levels of foliage symptoms had significantly lower levels of soil inoculum compared to lines with high levels of visible foliage symptoms. In a separate experiment, a set of superior recombinant inbred lines with consistently low levels of foliage symptoms was tested for soil inoculum reactions relative to control normalised yield loss. The in-crop P. medicaginis soil inoculum concentrations across genotypes were significantly and positively related to yield loss, indicating a partial resistance-tolerance spectrum. Disease incidence and the rankings for in-crop soil inoculum were correlated strongly to yield loss. These results indicate that soil inoculum reactions may be useful to identify genotypes with high levels of partial resistance.

Chickpea pathogen-resistance-rating correlates with responsiveness to an immune elicitor protein from Phytophthora medicaginis

Identifying elicitors of plant defence from soilborne plant pathogens can provide valuable knowledge about plant-microbe interactions and can be used as an efficient screening tool in breeding programs to identify pathogen resistant genotypes. Phytophthora medicaginis causes Phytophthora Root Rot (PRR) in a range of Fabaceae hosts including soybean and chickpea. The P. medicaginis:soybean pathosystem has been well studied and qualitative plant resistance loci identified, however less is known concerning the interaction with chickpea and only quantitative resistance has been identified to date. We hypothesized that P. medicaginis encodes elicitors that are perceived by chickpea and that the responsiveness of different genotypes to the elicitor(s) would vary with disease resistance. Using sequential purification, we successfully isolated proteinaceous fractions that contained elicitor molecule(s) from the mycelium of Phytophthora medicaginis but not from the culture supernatant. We were able to purify a ∼22 kDa protein from these fractions that induced three classical plant markers for elicitor perception: an average of 2.5x more ethylene than control treatment, deposition of callose, and an increase of 2.1x in phenylalanine ammonia-lyase activity. PmEip1 perception was found to elicit strong responses in other Fabaceae hosts, and that its activity in chickpea plants varied with the degree of PRR susceptibility whereby chickpea varieties with higher levels of PRR resistance were more responsive to PmEip1 than susceptible varieties. Using tandem mass spectrometry, we found that PmEip1 shared 87.6% homology to a glyceraldehyde-3-phosphate dehydrogenase from Phytophthora cactorum. Therefore, P. medicaginis produces an elicitor protein that is perceived by chickpea. This elicitor protein could be used as a means of screening for resistant genotypes in future chickpea breeding programs.

Chickpea Roots Undergoing Colonisation by Phytophthora medicaginis Exhibit Opposing Jasmonic Acid and Salicylic Acid Accumulation and Signalling Profiles to Leaf Hemibiotrophic Models.

Hemibiotrophic pathogens cause significant losses within agriculture, threatening the sustainability of food systems globally. These microbes colonise plant tissues in three phases: a biotrophic phase followed by a biotrophic-to-necrotrophic switch phase and ending with necrotrophy. Each of these phases is characterized by both common and discrete host transcriptional responses. Plant hormones play an important role in these phases, with foliar models showing that salicylic acid accumulates during the biotrophic phase and jasmonic acid/ethylene responses occur during the necrotrophic phase. The appropriateness of this model to plant roots has been challenged in recent years. The need to understand root responses to hemibiotrophic pathogens of agronomic importance necessitates further research. In this study, using the root hemibiotroph Phytophthora medicaginis, we define the duration of each phase of pathogenesis in Cicer arietinum (chickpea) roots. Using transcriptional profiling, we demonstrate that susceptible chickpea roots display some similarities in response to disease progression as previously documented in leaf plant–pathogen hemibiotrophic interactions. However, our transcriptomic results also show that chickpea roots do not conform to the phytohormone responses typically found in leaf colonisation by hemibiotrophs. We found that quantified levels of salicylic acid concentrations in root tissues decreased significantly during biotrophy while jasmonic acid concentrations were significantly induced. This study demonstrated that a wider spectrum of plant species should be investigated in the future to understand the physiological changes in plants during colonisation by soil-borne hemibiotrophic pathogens before we can better manage these economically important microbes.

Responsiveness to a Novel Elicitor from Phytophthora Medicaginis Increases with Pathogen Resistance Rating in Chickpea (Cicer Arietinum)

Responsiveness to a Novel Elicitor from Phytophthora Medicaginis Increases with Pathogen Resistance Rating in Chickpea (Cicer Arietinum)

Cause of Death: Phytophthora or Flood? Effects of Waterlogging on Phytophthora medicaginis and Resistance of Chickpea (Cicer arietinum)

Chickpea production in Australia is constrained by both waterlogging and the root disease Phytophthora root rot (PRR). Soil saturation is an important pre-condition for significant disease development for many soil-borne Phytophthora spp. In wet years, water can pool in low lying areas within a field, resulting in waterlogging, which, in the presence of PRR, can result in a significant yield loss for Australian chickpea varieties. In these circumstances, the specific cause of death is often difficult to discern, as the damage is rapid and the spread of PRR can be explosive in nature. The present study describes the impact of soil waterlogging on oxygen availability and the ability of P. medicaginis to infect chickpea plants. Late waterlogging in combination with PRR reduced the total plant biomass by an average of 94%; however, waterlogging alone accounted for 88% of this loss across three reference genotypes. Additional experiments found that under hypoxic conditions associated with waterlogging, P. medicaganis did not proliferate as determined by zoospore counts and DNA detection using qPCR. Consequently, minimizing waterlogging damage through breeding and agronomic practices should be a key priority for integrated disease management, as waterlogging alone results in plant stunting, yield loss and a reduced resistance to PRR.

Phenotyping for Waterlogging Tolerance as a Proxy for Phytophthora medicaginis Resistance in Chickpea

Phytophthora root rot (PRR) caused by the soilborne oomycete Phytophthora medicaginis is a significant constraint to chickpea (Cicer arietinum) production across the northern grains region of Australia. In flooded soil, which is conducive to PRR disease development, up to 70% yield loss can occur in the most resistant Australian cultivars. Incorporating waterlogging tolerance in soybean (Glycine max) has been shown to improve quantitative resistance to Phytophthora sojae. Root growth of three chickpea genotypes was assessed at the seedling stage under waterlogging, PRR, and the combination of these abiotic and biotic constraints. Levels of waterlogging tolerance in chickpea are inherently low, yet selected genotypes displayed variability in root traits linked to improved waterlogging tolerance. The PRR moderately susceptible chickpea cultivar Yorker and PRR very susceptible Rupali demonstrated an eightfold increase in early adventitious root growth from the epicotyl region under waterlogging stress, compared with the PRR resistant interspecific backcross genotype 04067-81-2-1-1 (C. echinospermum × C. arietinum*2). Selection for primary root depth, which was significantly greater in 04067-81-2-1-1 under waterlogging, appears to improve PRR resistance compared with root replacement traits. Soilborne Phytophthora spp. are reportedly attracted to branch sites and leached exudates. We propose that compromised root barriers at emergence sites of adventitious roots under waterlogging conditions hasten hyphal entry, potentially increasing susceptibility to PRR. Hence, screening for root depth and absence of adventitious root development under waterlogged conditions may offer a novel proxy phenotyping method for PRR resistance traits at early stages of chickpea breeding.

Order of microbial succession affects rhizobia-mediated biocontrol efforts against Phytophthora root rot

The management of soilborne root diseases in pulse crops is challenged by a limited range of resistance sources and often a complete absence of in-crop management options. Therefore, alternative management strategies need to be developed. We evaluated disease limiting interactions between the rhizobia species Mesorhizobium ciceri, and the oomycete pathogen Phytophthora medicaginis, which causes Phytophthora root rot (PRR) of chickpea (Cicer arietinum). For the PRR susceptible var. Sonali plants, post-pathogen M. ciceri inoculation significantly improved probability of plant survival when compared to P. medicaginis infected plants only pre-inoculated with M. ciceri (75 % versus 35 %, respectively). Potential mechanisms for these effects were investigated: rhizobia inoculation benefits to plant nodulation were not demonstrated, but the highest nodule N-fixation activity of P. medicaginis inoculated plants occurred for the post-pathogen M. ciceri treatment; rhizobia inoculation treatment did not reduce lesion development but certain combinations of microbial inoculation led to significant reduction in root growth. Microcosm studies, however, showed that the presence of M. ciceri reduced growth of P. medicaginis isolates. Putative chickpea disease resistance gene expression was evaluated using qPCR in var. Sonali roots. When var. Sonali plants were treated with M. ciceri post-P. medicaginis inoculation, the gene regulation in the plant host became more similar to PRR moderately resistant var. PBA HatTrick. These results suggest that M. ciceri application post P. medicaginis inoculation may improve plant survival by inducing defense responses similar to a PRR moderately resistant chickpea variety. Altogether, these results indicate that order of microbial succession can significantly affect PRR plant survial in susceptible chickpea under controlled conditions and improved plant survival effects are due to a number of different mechanisms including improved host nutrition, through direct inhibiton of pathogen growth, as well as host defense priming.

Phytophthora root rot of chickpea: inoculum concentration and seasonally dependent success for qPCR based predictions of disease and yield loss

Phytophthora root rot (PRR) caused by Phytophthora medicaginis (Pm) is an important disease of chickpea in Australia. There are limited control options, with avoiding planting chickpeas in paddocks with a high PRR risk a key management strategy. Currently, risk assessment is based solely on paddock history of PRR, without any measure of Pm inoculum. We developed a qPCR test to quantify Pm inoculum concentrations in soil and evaluated its ability to predict, prior to planting, PRR disease and yield loss in chickpea. The qPCR test was specific to Pm and did not cross react with other Phytophthora species found in Australian agricultural systems and was sensitive, being able to detect <1 Pm oospore/g soil. A field experiment showed no correlation between Pm DNA concentration at seeding and PRR development on the susceptible chickpea variety, Sonali. Field experiments with Yorker, a PRR moderately resistant variety, showed Pm DNA concentration at seeding was correlated with PRR development and grain yield, but relationships varied with season and soil moisture (2015, irrigated r2 0.752, dryland r2 0.532; 2017 irrigated r2 0.52, dryland r2 0.0005), however above average rainfall in 2016 was highly conducive to PRR and there was no relationship between Pm DNA concentrations at seeding and PRR development or yield. The qPCR could detect moderate to high levels of Pm inoculum, but not low levels capable of causing disease and yield loss in conducive seasons. The Pm qPCR test showed potential to support in-field diagnostics.

Effects of Mesorhizobium ciceri and Biochar on the Growth, Nodulation and Antifungal Activity Against Root Pathogenic Fungi in Chickpea (Cicer arietinum L.)

This study was conducted to investigate the effects of the inoculation of Mesorhizobium ciceri on the nodulation, growth and antagonistic expression against soil-borne fungal pathogens (Phytophthora medicaginis, Fusarium oxysporum and Fusarium solani) on Cicer arietinum L. or commonly known as chickpea grown in vermiculite medium amended with 5% green waste (GW) biochar. The combination of M. ciceri and biochar showed significant effect to chickpea compared with other treatments and control plants in terms of nodulation. The chickpea inoculated with M. ciceri and amended with biochar produced the highest nodule number with an average value of 110 nodules per plant and with an average nodule fresh weight of 57.90 mg per plant at 60 days harvest. The other treatments (M. ciceri only and biochar only) and the positive control (2 mM nitrate-treated plants) produced an average of 55, 65 and 15 nodules per plant with the corresponding average nodule weight of 39.5, 46.5 and 35.6 mg per plant, respectively, 60 days after seed sowing. The combined M. ciceri and biochar also enhanced the shoot length, and fresh and dry weights of chickpea. However, it was observed that the primary root length was shorter than the control but clusters of feeder roots were observed. The combination of M. ciceri and biochar also completely inhibited the colony development of all root pathogenic fungi of chickpea after three days of inoculation. Therefore, the inoculation of M. ciceri in vermiculite medium amended with green waste biochar enhanced the nodulation and growth conditions in chickpea as well as inhibited the growth of root pathogenic fungi P. medicaginis, F. oxysporum and F. solani.

Single plant selection for improving root rot disease (Phytophthora medicaginis) resistance in Chickpeas (Cicer arietinum L.)

The root rot caused by Phytophthora medicaginis is a major disease of chickpea in Australia. Grain yield loss of 50 to 70% due to the disease was noted in the farmers’ fields and in the experimental plots, respectively. To overcome the problem, resistant single plants were selected from the National Chickpea Multi Environment Trials (NCMET)—Stage 3 (S3) of NCMET-S1 to S3, which were conducted in an artificially infected phytophthora screening field nursery in the Hermitage Research Station, Queensland. The inheritance of resistance of these selected resistant single plants were tested in the next generation in three different trials, (1) at seedling stage in a shade house during the off-season, (2) as bulked single plants and (3) as individual single plants in the disease screening filed nursery during the next season. The results of the tests showed that many of the selected single plants had higher level of resistance and seed yield (P < 0.05) than the best resistant cultivars used as checks. The single plants with bidirectional expression of anthocyanin pigment, both at the shoot apex and at the base of stem, showed higher level of resistance than plants without it. The results also indicated certain level of heterozygosity-induced heterogeneity, which could cause higher levels of susceptibility, if the selected single plants were not screened further for the disease resistance in advanced generation/s. The genetics of resistance to PRR disease was confirmed as quantitative in nature.

Mapping resistance to Phytophthora root rot identifies independent loci from cultivated (Cicer arietinum L.) and wild (Cicer echinospermum P.H. Davis) chickpea.

Major QTL for Phytophthora root rot resistance have been identified in three mapping populations with independent sources of resistance contributed by C. echinospermum and C. arietinum. Phytophthora root rot (PRR) caused by the oomycete Phytophthora medicaginis is a major soil-borne disease of chickpea in Australia. With no economic in-crop control of PRR, a genetic approach to improve resistance is the most practical management option. Moderate field resistance has been incorporated in the cultivated C. arietinum variety, Yorker, and a higher level of resistance has been identified in a derivative of wild chickpea (C. echinospermum, interspecific breeding line 04067-81-2-1-1). These genotypes and two other released varieties were used to develop one intra-specific and two interspecific F6-derived recombinant inbred line mapping populations for genetic analysis of resistance. The Yorker × Genesis114 (YG), Rupali × 04067-81-2-1-1 (RB) and Yorker × 04067-81-2-1-1 (YB) populations were genotyped using genotyping-by-sequencing and phenotyped for PRR under three field environments with a mixture of 10 P. medicaginis isolates. Whole-genome QTL analysis identified major QTL QRBprrsi01, QYBprrsi01, QRBprrsi03 and QYBprrsi02 for PRR resistance on chromosomes 3 and 6, in RB and YB populations, respectively, with the resistance source derived from the wild Cicer species. QTL QYGprrsi02 and QYGprrsi03 were also identified on chromosomes 5 and 6 in YG population from C. arietinum. Aligning QTL regions to the corresponding chickpea reference genome suggested that the resistance source from C. arietinum and C. echinospermum may be different. The findings from this study provide tools for marker-assisted selection in chickpea breeding and information to assist the development of populations suitable for fine-mapping of resistance loci to determine the molecular basis for PRR resistance in chickpea.

Improved Phytophthora resistance in commercial chickpea (Cicer arietinum) varieties negatively impacts symbiotic gene signalling and symbiotic potential in some varieties.

Breeding disease-resistant varieties is one of the most effective and economical means to combat soilborne diseases in pulse crops. Commonalities between pathogenic and mutualistic microbe colonization strategies, however, raises the concern that reduced susceptibility to pathogens may simultaneously reduce colonization by beneficial microbes. We investigate here the degree of overlap in the transcriptional response of the Phytophthora medicaginis susceptible chickpea variety 'Sonali' to the early colonization stages of either Phytophthora, rhizobial bacteria or arbuscular mycorrhizal fungi. From a total of 6476 genes differentially expressed in Sonali roots during colonization by any of the microbes tested, 10.2% were regulated in a similar manner regardless of whether it was the pathogenic oomycete or a mutualistic microbe colonizing the roots. Of these genes, 49.7% were oppositely regulated under the same conditions in the moderately Phytophthora resistant chickpea variety 'PBA HatTrick'. Chickpea varieties with improved resistance to Phytophthora also displayed lower colonization by rhizobial bacteria and mycorrhizal fungi leading to an increased reliance on N and P from soil. Together, our results suggest that marker-based breeding in crops such as chickpea should be further investigated such that plant disease resistance can be tailored to a specific pathogen without affecting mutualistic plant:microbe interactions.

A Mineral Seed Coating for Control of Seedling Diseases of Alfalfa Suitable for Organic Production Systems.

Most alfalfa seed is treated with the fungicide mefenoxam (Apron XL) for control of soilborne seedling diseases caused by Phytophthora medicaginis and Pythium spp. However, Apron XL is not active against Aphanomyces euteiches, the causal agent of Aphanomyces root rot (ARR), an important component of the alfalfa seedling root rot complex. Moreover, Apron XL-treated seed cannot be used in organic production systems. A seed coating using aluminosilicate (natural zeolite) at a rate of 0.33 g of zeolite per gram of alfalfa seed was tested as an alfalfa seed treatment. Inoculated growth chamber trials were conducted to determine the percentage of seedlings protected from Phytophthora root rot (PRR) and ARR. The mineral seed coating resulted in significantly greater control of PRR, with a mean of 89% healthy seedlings (disease score of 1 or 2 on a 1-to-5 scale) compared with the Apron XL treatment, with a mean of 38% healthy seedlings, or the control treatment, with 15% healthy seedlings. The mineral seed coating also resulted in significantly greater protection against ARR, with 67% healthy seedlings compared with 3 and 2% healthy seedlings with the Apron XL and control treatments, respectively. The coated seed were used for in vitro assays with Pythium ultimum and P. paroecandrum to test for protection from seed rot and damping off. The mineral seed coating resulted in a significantly greater percentage of healthy seedlings compared with the Apron XL and control treatments. In growth chamber assays with naturally infested field soils with a range of disease pressure, the mineral seed coating resulted in a similar or greater percentage of healthy plants than the Apron XL treatment. The mineral coating had no effect on in vitro growth of Sinorhizobium meliloti, and nodule numbers were similar on roots from mineral-coated and untreated seed. These experiments indicate that the zeolite seed coating is a promising means of controlling seedling diseases in alfalfa production systems.

Expression of coordinately regulated defence response genes and analysis of their role in disease resistance in Medicago truncatula.

Microarray technology was used to identify the genes associated with disease defence responses in the model legume Medicago truncatula. Transcript profiles from M. truncatula cv. Jemalong genotype A17 leaves inoculated with Colletotrichum trifolii and Erysiphe pisi and roots infected with Phytophthora medicaginis were compared to identify the genes expressed in response to all three pathogens and genes unique to an interaction. The A17 genotype is resistant to C. trifolii and E. pisi, exhibiting a hypersensitive response after inoculation, and is moderately susceptible to P. medicaginis. Among the most strongly up-regulated genes in all three interactions were those encoding a hevein-like protein, thaumatin-like protein (TLP) and members of the pathogenesis response (PR)10 family. Transcripts of genes for enzymes in the phenylpropanoid pathway leading to the production of isoflavonoid phytoalexins increased dramatically in response to inoculation with the foliar pathogens. In P. medicaginis-inoculated roots, transcripts of genes in the phenylpropanoid pathway peaked at 5 days post-inoculation, when symptoms became visible. Transcript accumulation of three PR10 family members, a TLP and chalcone synthase (CHS) was assessed in M. truncatula genotype R108 plants. The R108 plants are resistant to C. trifolii and moderately susceptible to E. pisi and P. medicaginis. Transcript accumulation paralleled the stages of pathogen development. To evaluate the role of a TLP, a PR10 family member and CHS in disease resistance, transgenic R108 plants containing interfering RNA (RNAi) constructs were produced. Reduced expression of PR10 and TLP had no effect on the disease phenotype, whereas reduced expression of CHS resulted in increased susceptibility to necrotrophic pathogens.

Biocontrol of chickpea root rot using endophytic actinobacteria

Eleven actinobacterial strains were isolated from different plants, lentil (Lens esculentus), chickpea (Cicer arietinum L.), pea (Pisum sativum), faba bean (Vicia faba) and wheat (Triticum vulgare) from Paskerville, South Australia. Isolates were characterized and identified morphologically as well as using 16S ribosomal RNA gene sequencing. Of the actinobacteria tested, 72% produced siderophores, 33% were positive for cyanogens production, and 11% showed phosphate solubility. All isolates had antimicrobial activity against Phytophthora medicaginis, Pythium irregulare and Botrytis cinerea. In a greenhouse experiment, actinobacteria with the highest biocontrol capabilities were tested for their ability to control Phytophthora root rot on chickpea. Both Streptomyces sp. BSA25 and WRA1 successfully suppressed Phytophthora root rot when coinoculated with either Mesorhizobium ciceri WSM1666 or Kaiuroo 3. Streptomyces sp. BSA25 with either rhizobial strain enhanced vegetative growth of root (7–11 fold) and shoot dry weights (2–3 fold) compared to infected control, whereas Streptomyces sp. WRA1 increased root and shoot dry weights by 8- and 4-fold, respectively when inoculated with M. ciceri WSM1666. We suggest that careful selection of actinobacteria should be considered when coinoculated with beneficial microorganisms as plant symbionts.