Biocontrol traits of plant growth suppressive arbuscular mycorrhizal fungi against root rot in tomato caused by <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis>

Arbuscular mycorrhizal (AM) fungi known to cause plant growth depressions in tomato were examined for their biocontrol effects against root rot caused by Pythium aphanidermatum. The main hypothesis was that plant growth suppressive AM fungi would elicit a defence response in the host plant reducing Pythium root rot development. To test this hypothesis a fully factorial experiment was performed with AM fungi (Glomus intraradices, G. mosseae, G. claroideum or nonmycorrhizal), Pythium (± P. aphanidermatum) and harvest (7 and 14 days after pathogen inoculation (dapi)) as the main factors. Two weeks after AM fungi inoculation, roots were challenged with P. aphanidermatum. Variables evaluated at each harvest were root colonization levels of the interacting fungi, plant growth responses, and expression of a plant pathogenesis related protein gene (PR-1). All of the tested AM fungi caused marked growth suppressions, but did not affect PR-1 gene expression or the phosphorous concentration in the host plant. Plants singly inoculated with P. aphanidermatum had an increased PR-1 expression and phosphorous concentration. Among the AM fungi included in the study only G. intraradices reduced the pathogen root infection level, measured both in terms of Pythium ELISA and by recovery on selective media and only at the first harvest. Likewise, P. aphanidermatum root infection reduced colonization levels of G. intraradices, but not that of the two other AM fungi. In conclusion, plant growth suppressive AM fungi may offer plant beneficial traits in terms of biocontrol of root cortical pathogens.     

Heat shock-induced resistance in strawberry against crown rot fungus Colletotrichum gloeosporioides

This present study investigated the effects of heat shock treatments in strawberry seedlings against crown rot caused by Colletotrichum gloeosporioides. Heat shock treatment at 50 °C for 20 s reduced the disease index of strawberry crown rot and increased chitinase 2-1 gene expression as well as free salycilic acid accumulation. Heat shock treatment did not reduce mycelial growth of C. gloeosporioides. BIT(2-benzisothiazol-3(2H)-one,1,1-dioxide) treatment could not protect strawberry seedlings against crown rot disease. This finding suggests that heat shock induces strawberry resistance against Colletotrichum crown rot and assumes that mechanisms other than SAR probably mediated the protective effect of heat shock-induced resistance.     

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

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

Wilt and root rot of poinsettia caused by three high-temperature-tolerant Pythium species in ebb-and-flow irrigation systems

Poinsettia plants growing in ebb-and-flow irrigation systems developed wilting and root rot during the summer growing seasons of 2010 in Gifu Prefecture and 2011 in Aichi Prefecture. Pythium species were isolated from roots with rot symptoms. The isolates were identified as P. helicoides and P. myriotylum on the basis of morphological characteristics and sequence homologies in the rDNA internal transcribed spacer regions. In pathogenicity tests, these isolates caused severe wilting and root rot. This is the first report of poinsettia root rot disease caused by P. helicoides and P. myriotylum, although P. aphanidermatum was reported as a pathogen of poinsettia root rot. To better understand these diseases, we performed an epidemiological study of three high-temperature-tolerant Pythium species, P. aphanidermatum, P. helicoides and P. myriotylum. Disease incidence as a percentage of diseased plants was greatest at 35 °C for all three species. Disease severity using the rating scale of root rot was also highest at 35 °C, particularly with high zoospore inoculum densities (100.0 zoospores/mL). Although the disease incidence and severity were reduced at lower temperatures, the three Pythium species were able to cause disease at temperatures as low as 20 °C.     

Insights into the molecular mechanism of tolerance to carboxylic acid amide (CAA) fungicides in Pythium aphanidermatum

BACKGROUND: Tolerance to the oomycete‐specific carboxylic acid amide (CAA) fungicides is a poorly understood mechanism in Pythium species. The root‐rot and damping‐off causative agent Pythium aphanidermatum and the CAA fungicide mandipropamid (MPD) were used to investigate the molecular basis of CAA tolerance. RESULTS: Five genes putatively involved in carbohydrate synthesis were identified and characterised: one chitin synthase gene, PaChs, and four cellulose synthase genes PaCesA1 to PaCesA4, of which PaCesA3 encodes the MPD target enzyme. These genes were differentially expressed throughout the life cycle of P. aphanidermatum. Mycelium treated with MPD concentrations slightly affecting mycelial growth did not cause a change in PaCesA3 expression nor a strong upregulation of PaCesA homologues. The high tolerance level of P. aphanidermatum and the lack of PaCesA upregulation imply that MPD tolerance is the result of a specific amino acid configuration in the cellulose synthase 3 (CesA3) target enzyme. Indeed, P. aphanidermatum displays the amino acid L1109 which is also associated with MPD resistance in artificial mutants of Phytophthora species. CONCLUSION: It is concluded that MPD tolerance in P. aphanidermatum is not caused by compensatory mechanisms but most likely by an inherent target‐site configuration in PaCesA3 that hinders MPD binding to the enzyme pocket. Copyright © 2012 Society of Chemical Industry     

Plant growth promoting rhizobacteria (PGPR) and entomopathogenic fungus bioformulation enhance the expression of defense enzymes and pathogenesis-related proteins in groundnut plants against leafminer insect and collar rot pathogen

The bioformulation of Pseudomonas fluorescens (Pf1 and TDK1) and Beauveria bassiana (B2) strains was evaluated individually and in combinations with and without chitin for their efficacy against leafminer insect and collar rot disease and the effect of the interaction between Pseudomonas, Beauveria and groundnut leafminer insect and collar rot pathogen in the expression of defense enzymes and pathogenesis-related proteins (PR-proteins) in groundnut. Among the various bioformulations, B2 + TDK1 + Pf1 (amended with or without chitin) formulation significantly reduced the incidence of leafminer and collar rot disease when compared to untreated control. A significant increase in the enzymatic activity of phenylalanine ammonia-lyase, peroxidase, polyphenol oxidase, chitinase, β-1,3-glucanase, superoxide dismutase, catalase, lipoxygenase, and phenolics in groundnut plants treated with B2 + TDK1 + Pf1 bioformulation (amended with or without chitin) and challenge inoculated with Aproaerema modicella and Sclerotium rolfsii. Native gel electrophoresis also revealed the expression of more isoforms of pathogenesis-related proteins and other defense enzymes viz., polyphenol oxidase and superoxide dismutase in plants treated with B2 + TDK1 + Pf1 mixture challenged with A. modicella and S. rolfsii. The present study reveals that sustained and timely induction and accumulation of these defense enzymes and PR-proteins enhance the resistance in groundnut against leafminer insect and collar rot disease.     

Defense enzymes induced in cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum

Root and crown rot of cucumber caused by Pythium aphanidermatum can be suppressed by various rhizobacteria or PGPR (plant growth-promoting rhizobacteria). When cucumber roots were treated with Pseudomonas corrugata 13 or Pseudomonas aureofaciens 63–28, phenylalanine ammonia-lyase (PAL) activity was stimulated in root tissues in 2 days and this activated accumulation lasted for 16 days after bacterization. Peroxidase (PO) and polyphenol oxidase (PPO) activities were increased in roots 2–5 days after bacterization with P. corrugata strain 13. After bacterized cucumber roots were challenged with P. aphanidermatum, the enzyme activities of PAL, PO and PPO increased as the disease developed on the roots. These accumulations peaked 4–6 days after pathogen inoculation. A split root system demonstrated that the three enzymes could be systemically induced by the Pseudomonas strains 63–28 and 13, as well as P. aphanidermatum. Furthermore, isoperoxidase native PAGE (polyacrylamide gel electrophoresis) analysis indicated that the peroxidase isomer forms in cucumber roots induced by rhizobacteria were different from that in roots infected with P. aphanidermatum. These results suggest that the plant defense enzymes could be stimulated in cucumber roots which have been colonized by non-pathogenic rhizobacteria or in a compatible interaction between cucumber and P. aphanidermatum. The mechanisms of PO activation by the rhizobacteria may be different from those of pathogen infection.     

Pythium rot of Chinese cabbage (Brassica rapa L. subsp. pekinensis) caused by Pythium aphanidermatum

Severe rot was found at the base of leaves and stems of Chinese cabbage (Brassica rapa L. subsp. pekinensis) in Ibaraki Prefecture every year in early September from 2002 through 2004. The causal fungus was identified as Pythium aphanidermatum (Edson) Fitzpatrick. This is the first report of P. aphanidermatum on Chinese cabbage. A similar disease of Chinese cabbage caused by P. ultimum Trow var. ultimum is known as Pythium rot. We propose adding P. aphanidermatum as a new pathogen of this disease.     

Identification and characterization of differentially expressed novel miRNAs (21–24 nt) in a Macrophomina phaseolina resistant RIL line of jute (Corchorus capsularis L.)

A RIL population of jute developed by crossing one resistant accession CIM 036 and a susceptible variety JRC 412 was used to identify novel defence related miRNAs activated upon challenged inoculation with stem rot pathogen Macrophomina phaseolina. About a total of 15.7 million reads were generated from the resistant line with the read length of 1 × 50 bp. Here, we identified nine mature novel microRNAs which passed Minimum Free Energy (MFE Kcal/mol) criteria. Target site and secondary structure were predicted and most of them showed ubiquitination and selective autophagy activity with high expression value. Five novel miRNAs viz. Candidate_41, Candidate_9, Candidate_66, Candidate_65 and Candidate_8 had free energy less than −25 kcal/mol. Known microRNAs viz. miR-845b and miR-166 superfamily are abundantly expressed with high expression value. The sequence of jute miR-845b superfamily is identical to that of Arabidopsis thaliana except at 18th position, but unlike in A. thaliana it targets the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. In-silico analysis suggested that miR-845b and miR-166 superfamily provided NBS-LRR and ROS mediated defence and subsequently expression of novel microRNAs with selective autophagy activity enabled multi-layered defence cascade against M. phaseolina in jute.     

Overexpression of an nsLTPs-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases of oilseed rape worldwide and leads to considerable yield losses. In this study, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into oilseed rape (Zhongyou 821) by Agrobacterium-mediated transformation. In vitro experiments revealed that the mycelial growth of S. sclerotiorum was significantly inhibited when supplied with crude leaf extracts from transgenic oilseed rape plants overexpressing LJAMP2. Furthermore, in vivo studies showed that transgenic LJAMP2 plants had enhanced resistance to S. sclerotiorum. Semi-quantitative RT-PCR analysis showed that the LJAMP2 gene was transcribed in all transformed plants. In addition, we also found that overexpression of LJAMP2 in transgenic plants caused constitutive activation of the defense-related gene PR-1 and an increase of H₂O₂ production, but did not enhance PDF1.2 expression. Our results suggest that constitutive expression of the LJAMP2 gene from motherwort seeds might be exploited to improve the resistance of oilseed rape against S. sclerotiorum.     

Specific detection of<Emphasis Type="Italic">Pythium aphanidermatum</Emphasis> from hydroponic nutrient solution by booster PCR with DNA primers developed from mitochondrial DNA

Pythium aphanidermatum causes damping-off and root rot of vegetable crops in hydroponic systems. A DNA probe was isolated and modified from a library ofHindIII-digested mitochondrial DNA ofP. aphanidermatum that strongly hybridized to DNA ofP. aphanidermatum and weakly hybridized to DNA ofPythium deliense. Cross-hybridizing sequences were absent from DNA of plants and other related fungi. The probe detected as little as 5 ng ofP. aphanidermatum DNA and 250 ng ofP. deliense DNA in slot-blot assays.P. aphanidermatum was detected by a hybridization assay of total DNA extracted directly from infected roots. A pair of oligonucleotide primers P1 and RP2, which allowed amplification of a specific 0.65 kb DNA fragment ofP. aphanidermatum using polymerase chain reaction (PCR), was designed from a specific DNA probe. Specific amplification of this fragment fromP. aphanidermatum was highly sensitive, detecting template DNA as low as 0.1 pg total DNA by booster PCR. Specific booster PCR amplification using P1 and RP2 was successful in detectingP. aphanidermatum in naturally infected nutrient solution and roots of vegetables in a field hydroponic system. http://www.phytoparasitica.org posting Sept. 22, 2002.     

Tomato partial resistance to Rhizoctonia solani involves antioxidative defense mechanisms

The resistance interactions of four tomato cultivars to five Rhizoctonia solani isolates, causing foot rot, were investigated. Priming H₂O₂ accumulation, peroxidase and ascorbate peroxidase activity was observed in CH Falat, as a partially resistant cultivar. Maximum peroxidase activity in CH Falat was observed at 24 h post inoculation (hpi) which was earlier than maximum phenolics accumulation at 72 hpi. In addition, application of peroxidase inhibitor reduced phenolics level. Therefore, peroxidase might be involved in phenolics production, as an effective resistance mechanism in our pathosystem.     

Identification of isolate‐specific resistance QTLs to phytophthora root rot using an intraspecific recombinant inbred line population of pepper (Capsicum annuum)

Quantitative trait loci (QTL) for resistance to phytophthora root rot caused by Phytophthora capsici were investigated using two Korean P. capsici isolates and 126 F₈ recombinant inbred lines derived from a cross of Capsicum annuum line YCM334 (resistant parent) and local cv. Tean (susceptible parent). The experimental design was a split plot with two replications. Highly significant effects of pathogen isolate, plant genotype, and genotype × isolate were detected. QTL mapping was performed using a genetic linkage map covering 1486·6 cM of the pepper genome, and consisted of 249 markers including 136 AFLPs (Amplified Fragment Length Polymorphisms), 112 SSRs (Simple Sequence Repeats) and one CAPS (Cleaved Amplified Polymorphic Sequence). Fifteen QTLs were detected on chromosomes 5 (P5), 10 (P10), 11 (P11), Pb and Pc using two data processing methods: percentage of wilted plants (PWP) and relative area under the disease progress curves (RAUDPC). The phenotypic variation explained by each QTL (R²) ranged from 6·0% to 48·2%. Seven QTLs were common to resistance for the two isolates on chromosome 5 (P5); six were isolate‐specific for isolate 09‐051 on chromosomes 10 (P10) and Pc, and two for isolate 07‐127 on chromosomes 11 (P11) and Pb. The QTLs in common with the major effect on the resistance for two isolates explained 20·0–48·2% of phenotypic variation. The isolate‐specific QTLs explained 6·0–17·4% of phenotypic variation. The result confirms a gene‐for‐gene relationship between C. annuum and P. capsici for root rot resistance.     

Induction of disease resistance against Botrytis cinerea by heat shock treatment in melon (Cucumis melo L.)

The present study investigated resistance against Botrytis cinerea after heat shock treatment in melon plants. Heat shock at 50 °C for 20 s 0–24 h before inoculation resulted in maximal B. cinerea symptom reduction and peroxidase gene expression, which peaked 12 and 72 h post-treatment and decreased 24–48 h post-treatment, suggesting pathogenesis-related protein expression priming. Hot water dipping did not directly inhibit mycelia growth. Plants treated with 2-benzisothiazol-3(2H)-one 1,1-dioxide, which induces systemic acquired resistance, demonstrated higher peroxidase gene expression but no B. cinerea resistance, indicating possible involvement of additional novel mechanisms in heat shock-activated resistance of melon against B. cinerea.     

Production of hydrogen peroxide and expression of ROS‐generating genes in peach flower petals in response to host and non‐host fungal pathogens

Reactive oxygen species (ROS) play dual roles in plant–microbe interactions in that they can either stimulate host resistance or enhance pathogen virulence. Innate resistance in peach (Prunus persica) to the brown rot fungal pathogen Monilinia fructicola is very limited, and knowledge of the mechanism of virulence is rudimentary. In this study, production of hydrogen peroxide, a major component of ROS, was determined in peach flower petals in response to M. fructicola (a host pathogen) and Penicillium digitatum (a non‐host pathogen). Monilinia fructicola was able to infect flower petals while P. digitatum was not. During the host‐specific interaction, M. fructicola induced hydrogen peroxide accumulation in flower petals. Application of exogenous antioxidants significantly reduced hydrogen peroxide accumulation as well as the incidence of brown rot disease. Application of M. fructicola spores to the surface of intact flower petals induced gene expression and increased enzyme activity of NADPH oxidase and cell wall peroxidase in host tissues, resulting in the production of hydrogen peroxide. Petals inoculated with M. fructicola exhibited high levels of protein carbonylation and lipid peroxidation. No significant response in gene expression, enzyme activity or hydrogen peroxide levels was observed in peach flower petals treated with P. digitatum. These results suggest that M. fructicola, as with other necrotrophic fungi, uses the strong oxidative response as part of a virulence mechanism.