Interaction of proteinases secreted by the fungal plant pathogen Rhizoctonia solani with natural proteinase inhibitors produced by plants

The fungal plant pathogen Rhizoctonia solani Kuhn. grown in a medium containing thermostable potato tuber proteins produced proteinases active at moderately alkaline pH values. Electrophoretic analysis in polyacrylamide gel with SDS and copolymerized gelatin showed that the extracellular proteinase complex contained four components that differed in molecular weight. Studies on the action of the exoenzymes on various synthetic substrates indicated that the culture liquid of R. solani contained mainly trypsin-like proteinases. The exoproteinase activity was virtually completely suppressed by trypsin inhibitor proteins isolated from potato tubers and seeds of various legume species. The results suggest that the extracellular proteinases produced by R. solani play a significant role in attacking plant tissue, and natural inhibitors contribute to the protection of Solanaceae and Leguminosae from this fungal pathogen.     

Inactivation of phytotoxin produced by the rice sheath blight pathogen Rhizoctonia solani

The rice sheath blight pathogen, Rhizoctonia solani, produces a toxin designated as RS-toxin, a carbohydrate compound containing mainly alpha-glucose and mannose. Different microflora were tested for RS-toxin inactivation. Isolates of Trichoderma viride inactivated this toxin when it was provided as the sole food source, and these isolates reduced the severity of toxin-induced symptoms and electrolyte leakage from rice cells. The best-performing isolate, TvMNT7, produced two extracellular proteins of 110 and 17 kDa. The high molecular mass protein was shown to have alpha-glucosidase activity. The purified 110 kDa protein was able to reduce RS-toxin activity.     

Greek indigenous streptomycetes as biocontrol agents against the soil‐borne fungal plant pathogen Rhizoctonia solani

Aims

To examine the biocontrol potential of multiactive Greek indigenous Streptomyces isolates carrying antifungal activity against Rhizoctonia solani that causes damping‐off symptoms on beans.

Methods and Results

A total of 605 Streptomyces isolates originated from 12 diverse Greek habitats were screened for antifungal activity against R. solani DSM843. Almost one‐third of the isolates proved to be antagonistic against the fungus. From the above isolates, six were selected due to their higher antifungal activity, identified by analysing their 16S rRNA gene sequence and studied further. The obtained data showed the following: firstly, the isolates ACTA1383 and ACTA1557 exhibited the highest antagonistic activity, and therefore, they were selected for in vivo experiments using bean seeds as target; secondly, in solid and liquid culture experiments under optimum antagonistic conditions, the medium extracts from the isolates OL80, ACTA1523, ACTA1551 and ACTA1522 suppressed the growth of the fungal mycelium, while extracts from ACTA 1383 and ACTA1557 did not show any activity.

Conclusions

These results corresponded important indications for the utility of two Greek indigenous Streptomyces isolates (ACTA1557 and ACTA1383) for the protection of the bean crops from R. solani damping‐off symptoms, while four of them (isolates OL80, ACTA1523, ACTA1551 and ACTA1522) seem to be promising producers of antifungal metabolites.

Significance and Impact of the Study

This is the first study on the biocontrol of R. solani using multiactive Streptomyces isolates originated from ecophysiologically special Greek habitats. Our study provides basic information to further explore managing strategies to control this critical disease.     

Early induction of the Arabidopsis GSTF8 promoter by specific strains of the fungal pathogen Rhizoctonia solani

The Arabidopsis glutathione S-transferase GSTF8 promoter directs root-specific responses to stress. In this study, the response of this promoter to plant infection with Rhizoctonia solani was investigated using a luciferase reporter system. Arabidopsis seedlings harboring the GSTF8:luciferase construct were monitored in vivo for bioluminescence following infection with R. solani. Although the reporter gene was induced in infected roots, the response differed markedly between R. solani strains and was not observed with aggressive strains that caused death of the seedlings. The three strains tested in detail progressed through typical stages of infection, but ZG1-1 induced the GSTF8 promoter in most seedlings, ZG3 induced it in approximately 25% of seedlings, and ZG5 caused little response. Induction of specific root segments occurred early in the infection process in root regions with very limited mycelium visible. In root segments with substantial mycelium, GSTF8 promoter activity no longer was observed. Induction by ZG1-1 also was observed in plants harboring a tetramer of the ocs element from the GSTF8 promoter, suggesting that this element helps mediate the response. Crossing GSTF8:luciferase plants with plants harboring an Nah-G construct that degrades salicylic acid did not abolish the response, indicating that the GSTF8 promoter response to R. solani may be mediated by signals other than salicylic acid.     

Interaction between Meloidogyne incognita and Rhizoctonia solani on green beans

The interaction between Meloidogyne incognita (race 2) and Rhizoctonia solani (AG 4) in a root rot disease complex of green beans (Phaseolus vulgaris) was examined in a greenhouse pot experiment. Three week-old seedlings (cv. Contender) were inoculated with the nematode and/or the fungus in different combinations and sequences. Two months after last nematode inoculation, the test was terminated and data were recorded. The synchronized inoculation by both pathogens (N + F) increased the index of Rhizoctonia root rot and the number of root galls; and suppressed plant growth, compared to controls. However, the severity of root rot and suppression of plant growth were greater and more evident when inoculation by the nematode preceded the fungus (N → F) by two weeks. Nematode reproduction (eggs/g root) was adversely affected by the presence of the fungus except by the synchronized inoculation. When inoculation by nematode preceded the fungus, plant growth was severely suppressed and roots were highly damaged and rotted leading to a decrease of root galls and eggs.     

Highly polymorphic microsatellite loci in the rice- and maize-infecting fungal pathogen Rhizoctonia solani anastomosis group 1 IA

Ten polymorphic microsatellite loci were isolated and characterized from the rice- and maize-infecting Basidiomycete fungus Rhizoctonia solani anastomosis group AG-1 IA. All loci were polymorphic in two populations from Louisiana in USA and Venezuela. The total number of alleles per locus ranged from four to eight. All 10 loci were also useful for genotyping soybean-infecting R. solani AG-1 isolates from Brazil and USA. One locus, TC06, amplified across two other AG groups representing different species, showing species-specific repeat length polymorphism. This marker suite will be used to determine the global population structure of this important pathogenic fungus.     

Mungbean plants expressing BjNPR1 exhibit enhanced resistance against the seedling rot pathogen, Rhizoctonia solani

Mungbean, Vigna radiata (L.) Wilczek is an important pulse crop that is widely cultivated in semi- arid tropics. The crop is attacked by various soil-borne pathogens like Rhizoctonia solani, which causes dry rot disease and seriously affects its productivity. Earlier we characterized the non-expressor of pathogenesis related gene-1(BjNPR1) of mustard, Brassica juncea, the counterpart of AtNPR1 of Arabidopsis thaliana. Here, we transformed mungbean with BjNPR1 via Agrobacterium tumefaciens. Because of the recalcitrant nature of mungbean, the effect of some factors like Agrobacterium tumefaciens strains (GV2260 and LBA4404), pH, l-cysteine and tobacco leaf extract was tested in transformation. The transgenic status of 15 plants was confirmed by PCR using primers for nptII. The independent integration of T-DNA in transgenic plants was analyzed by Southern hybridization with an nptII probe and the expression of BjNPR1 was confirmed by RT–PCR. Some of the T₀ plants were selected for detached leaf anti-fungal bioassay using the fungus Rhizoctonia solani, which showed moderate to high level of resistance depending on the level of expression of BjNPR1. The seedling bioassay of transgenic T₂ plants indicated resistance against dry rot disease caused by R. solani.     

Modifications of plant cell activities by polypeptides secreted by Eutypa lata, a vineyard fungal pathogen

Eutypa dieback is a devastating disease induced in vineyards by the fungal pathogen Eutypa lata . The fungus colonizes the xylem tissues of trunk and cordons but is never found in the annual canes. Nevertheless, dwarfed shoots and leaf necrosis observed in diseased plants indicate that a necrotic signal can spread at a distance from the infected area. Eutypine, a small cyclic molecule, and related compounds have been postulated as the toxins inducing these symptoms. In this work, we evidence that E. lata secreted other metabolites of polypeptidic nature which induced toxic effects on canes and leaves of vines, and on leaves of other plant materials. The polypeptide fraction (PF) isolated from culture medium of mycelium induced transitory H⁺ fluxes and membrane depolarization of plant cells. Complementary assays with plasma membrane vesicles (PMV) showed that H⁺-ATPase is a primary site of action as indicated by inhibition of the enzyme activity and increase of H⁺ conductance of plasma membrane. The toxic effect was also obvious on respiration and photosynthesis. All these impairments led to a hindering in cell energetics and, as a consequence, to an inhibition of uptake of assimilates. Treatment with PF also triggered biological events, characteristics of elicitation as suggested by the early responses on cell membrane described above, the activation of NADPH oxidase and the activation of Phenylalanine ammonia lyase (PAL).     

Tolerance to the fungal pathogen Rhizoctonia solani AG4 of transgenic tobacco expressing the maize ribosome-inactivating protein b-32

The maize b-32 protein is a functional ribosome-inactivating protein (RIP), inhibiting in vitro translation in the cell-free reticulocyte-derived system and having specific N-glycosidase activity on 28S rRNA. Previous results indicated that opaque-2 (o2) mutant kernels, lacking b-32, show an increased susceptibility to fungal attack and insect feeding and that ectopic expression in plants of a barley and a pokeweed RIP leads to increased tolerance to fungal and viral infection. This prompted us to test whether b-32 might functi on as a protectant against pathogens. The b32.66 cDNA clone under the control of the potato wun1 gene promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Out of 23 kanamycin resistant regenerated shoots, 16 contained a PCR fragment of the corrrect size spanning the boundary between the promoter used and the coding region of the b-32 gene. Eight independently transformed tobacco lines were randomly chosen for protein analysis: all of them expressed b-32 protein. The data presented indicate that transgenic tobacco plants expressing b-32 show an increased tolerance against infection by the soil-borne fungal pathogen Rhizoctonia solani Kuhn     

Highly polymorphic in silico-derived microsatellite loci in the potato-infecting fungal pathogen Rhizoctonia solani anastomosis group 3 from the Colombian Andes

Fourteen polymorphic microsatellite DNA markers derived from the draft genome sequence of Rhizoctonia solani anastomosis group 3 (AG-3), strain Rhs 1AP, were designed and characterized from the potato-infecting soil fungus R. solani AG-3. All loci were polymorphic in two field populations collected from Solanum tuberosum and S. phureja in the Colombian Andes. The total number of alleles per locus ranged from two to seven, while gene diversity (expected heterozygosity) varied from 0.11 to 0.81. Considering the variable levels of genetic diversity observed, these markers should be useful for population genetic analyses of this important dikaryotic fungal pathogen on a global scale.     

Serine proteinase inhibitors from nematodes and the arms race between host and pathogen

Serine proteinase inhibitors are encoded by a large gene family of long evolutionary standing. Recent discoveries of parasite proteins that inhibit human serine proteinases, together with the complete genomic sequence from Caenorhabditis elegans, have provided a set of new serine proteinase inhibitors from more primitive metazoan animals such as nematodes. The structural features (e.g. reactive centre residues), gene organization (including intron arrangements) and inhibitory function and targets (e.g. inflammatory and coagulation pathway proteinase) all contribute important new insights into proteinase inhibitor evolution. Some parasite products have evolved that block enzymes in the mammalian host, but the human host responds with a significant immune response to the parasite inhibitors. Thus, infection produces a finely balanced conflict between host and pathogen at the molecular level, and this might have accelerated the evolution of these proteins in parasitic species as well as their hosts.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Distinctively variable sequence-based nuclear DNA markers for multilocus phylogeography of the soybean- and rice-infecting fungal pathogen Rhizoctonia solani AG-1 IA

A series of multilocus sequence-based nuclear DNA markers was developed to infer the phylogeographical history of the Basidiomycetous fungal pathogen Rhizoctonia solani AG-1 IA infecting rice and soybean worldwide. The strategy was based on sequencing of cloned genomic DNA fragments (previously used as RFLP probes) and subsequent screening of fungal isolates to detect single nucleotide polymorphisms (SNPs). Ten primer pairs were designed based on these sequences, which resulted in PCR amplification of 200-320 bp size products and polymorphic sequences in all markers analyzed. By direct sequencing we identified both homokaryon and heterokaryon (i.e. dikaryon) isolates at each marker. Cloning the PCR products effectively estimated the allelic phase from heterokaryotic isolates. Information content varied among markers from 0.5 to 5.9 mutations per 100 bp. Thus, the former RFLP codominant probes were successfully converted into six distinctively variable sequence-based nuclear DNA markers. Rather than discarding low polymorphism loci, the combination of these distinctively variable anonymous nuclear markers would constitute an asset for the unbiased estimate of the phylogeographical parameters such as population sizes and divergent times, providing a more reliable species history that shaped the current population structure of R. solani AG-1 IA.     

Atypical Kunitz-type serine proteinase inhibitors produced by the ruminant placenta

Recently, an unusual family of genes was identified with expression confined to the trophoblast of ruminant ungulate species. The members of this family (the trophoblast Kunitz domain proteins, or TKDPs) are characterized by the presence of one or more similar, approximately 80-residue repeat sequences placed ahead of a Kunitz serine proteinase-inhibitor domain. To examine the specificity of the Kunitz moiety, the Kunitz domains of selected TKDPs and a control Kunitz protein, bovine pancreatic trypsin inhibitor (BPTI), were produced as glutathione S-transferase fusions, and their abilities to inhibit six serine proteinases were examined. Circular dichroism spectroscopy confirmed that the Kunitz fold was intact. Three of the TKDPs had unusual residues at their P1 "warhead" (ovine TKDP-1, Asn; bovine TKDP-3, Thr; and bovine TKDP-5, Ile) and exhibited no measurable inhibitory activity toward any of the proteinases. Three (ovine TKDP-3, bovine TKDP-3, and bovine TKDP-4) lacked the conserved cysteines at residues 14 and 38 that form one of the highly conserved disulfide bonds that are structurally important in all known mammalian Kunitz proteins. Ovine TKDP-3 and bovine TKDP-4 had P1 lysines and inhibited trypsin and plasmin with K(i) values only approximately 10-fold higher than that of BPTI. Bovine TKDP-2 had a P1 lysine and the three conserved disulfides, but it possessed an unusual residue (Asp) at P2. It exhibited no inhibitory activity. These data suggest that the function of the TKDP, like certain Kunitz proteins found in snake venoms, may not be in proteinase inhibition.     

Detection and characterization of protease secreted by the plant pathogen Xylella fastidiosa

Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a copolymerized substrate. Three major protein bands were produced by the citrus strain with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9,713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) activity gel indicated that the protease of Xylella fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30 degrees C with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and beta-1,3-glucanase activities were also detected in cultures of Xylella fastidiosa (citrus). From these results, it is suggested that proteases produced by strains of Xylella fastidiosa from citrus and grape, belong to the serine- and metallo-protease group, respectively.