Chitosan as a Component of Pea-Fusarium solani Interactions

Chitosan, a polymer of β-1,4-linked glucosamine residues with a strong affinity for DNA, was implicated in the pea pod-Fusarium solani interaction as an elicitor of phytoalexin production, an inhibitor of fungal growth and a chemical which can protect pea tissue from infection by F. solani f. sp. pisi. Purified Fusarium fungal cell walls can elicit phytoalexin production in pea pod tissue. Enzymes from acetone powders of pea tissue release eliciting components from the F. solani f. sp. phaseoli cell walls. Hydrochloric acid-hydrolyzed F. solani cell walls are about 20% glucosamine. The actual chitosan content of F. solani cell walls is about 1%. However, chitosan assays and histochemical observations indicate that chitosan content of F. solani spores and adjacent pea cells increases following inoculation. Dormant F. solani spores also accumulate chitosan. Concentrations of nitrous acid-cleaved chitosan as low as 0.9 microgram per milliliter and 3 micrograms per milliliter elicit phytoalexin induction and inhibit germination of F. solani macroconidia, respectively. When chitosan is applied to pea pod tissue with or prior to F. solani f. sp. pisi, the tissue is protected from infection.     

Localization of Fungal Components in the Pea-Fusarium Interaction Detected Immunochemically with Anti-chitosan and Anti-fungal Cell Wall Antisera

Antisera specific for purified cell walls of Fusarium solani f. sp. pisi and phaseoli and of shrimp shell chitosan were utilized as immunochemical probes to determine the location of fungal components in the pea-Fusarium interaction.     

Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase

Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.     

Identification of 1 a-hydroxy phaseollone,a phaseollin metabolite produced by Fusarium solani

A structure for the phaseollin metabolite of Fusarium solani f. sp phaseoli has been proposed and assigned the name 1 a-hydroxyphaseollone.     

Ethylene: Symptom, Not Signal for the Induction of Chitinase and beta-1,3-Glucanase in Pea Pods by Pathogens and Elicitors

Infection of immature pea pods with Fusarium solani f.sp. phaseoli (a non-pathogen of peas) or f.sp. pisi (a pea pathogen) resulted in induction of chitinase and β-1,3-glucanase. Within 30 hours, activities of the two enzymes increased 9-fold and 4-fold, respectively. Chitinase and β-1,3-glucanase were also induced by autoclaved spores of the two F. solani strains and by the known elicitors of phytoalexins in pea pods, cadmium ions, actinomycin D, and chitosan. Furthermore, exogenously applied ethylene caused an increase of chitinase and β-1,3-glucanase in uninfected pods. Fungal infection or treatment with elicitors strongly increased ethylene production by immature pea pods. Infected or elicitor-treated pea pods were incubated with aminoethoxyvinylglycine, a specific inhibitor of ethylene biosynthesis. This lowered stress ethylene production to or below the level of uninfected controls; however, chitinase and β-1,3-glucanase were still strongly induced. It is concluded that ethylene and fungal infection or elicitors are separate, independent signals for the induction of chitinase and β-1,3-glucanase.     

Detoxification of phaseollidin by Fusarium solani f.sp. Phaseoli

The phytoalexin phaseollidin is transformed into phaseollidin hydrate by liquid mycelial cultures and cell-free culture filtrates of Fusarium solani f.sp. phaseoli. The antifungal activity of the hydrate is much less than that of the original phytoalexin.     

Developmental Regulation of Susceptibility and Tolerance to Fusarium Root Rot in Beans

The soil-borne fungus, Fusarium solani f. sp. phaseoli, attacks roots and hypocotyls of bean (Phaseolus vulgaris) plants causing a devastating disease called root and foot rot. In a study of the host-pathogen relationship it was found that young bean roots, with the radicle just emerging, were highly tolerant to the pathogen, whereas older bean seedlings, with a fully developed root system, were completely susceptible. Investigations by low-temperature scanning electron microscopy demonstrated that significantly fewer spores and hyphae were present on the root surface of young bean seedlings as compared to older ones. A similar pattern of attachment was found when bean roots were inoculated with spores of F. solani f. sp. pisi, a related pathogen causing disease on peas but not on beans. Light microscopic studies showed that F. solani f. sp. pisi did not penetrate the root but rapidly formed thick-walled resting spores on the root surface. F. solani f. sp. phaseoli on the other hand quickly penetrated the root and formed an extensive network of fungal hyphae. These results demonstrate that the ability of fungal propagules to adhere to and to penetrate host tissues are two distinct processes. Furthermore, the data indicate that young bean roots lack a surface component necessary for attachment of fungal spores which may help explain their tolerance to Fusarium root rot.     

Metabolism of the phytoalexins medicarpin and maackiain by Fusarium solani

Non-inhibitory concentrations of the pterocarpan phytoalexin medicarpin were completely metabolized by isolates of Fusarium solani f. sp. pisi, f. sp. cucurbitae, f. sp. phaseoli and two other F. solani isolates genetically related to f. sp. pisi during 24 hr of growth in liquid medium. The major metabolic products accumulated without significant further degradation. Medicarpin was modified at one of three adjacent carbon atoms to form either an isoflavanone derivative, a 1a-hydroxydienone derivative or 6a-hydroxymedicarpin. Whereas each isolate degraded medicarpin to one or more metabolises, the isolates varied as to which metabolise they produced. Maackiain, another pterocarpan phytoalexin, was also metabolized by all the isolates to products analogous to those formed from medicarpin. The ability to metabolize medicarpin and maackiain was not always associated with the ability to metabolize pisatin and phaseollin, two other pterocarpan phytoalexins that were degraded by several of the isolates. Tolerance of medicarpin and maackiain was similarly not always associated with tolerance to pisatin.     

Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding,fungal infection and the elicitor chitosan

The fungicidal class I endochitinases (E.C.3.3.1.14, chitinase) are associated with the biochemical defense of plants against potential pathogens. We isolated and sequenced a genomic clone, DAH53, corresponding to a class I basic endochitinase gene in pea, Chil. The predicted amino acid sequence of this chitinase contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants. The pea genome contains one gene corresponding to the chitinase DAH53 probe. Chitinase RNA accumulation was observed in pea pods within 2 to 4 h after inoculation with the incompatible fungal strain Fusarium solani f. sp. phaseoli, the compatible strain F. solani f.sp. pisi, or the elicitor chitosan. The RNA accumulation was high in the basal region (lower stem and root) of both fungus challenged and wounded pea seedlings. The sustained high levels of chitinase mRNA expression may contribute to later stages of pea's non-host resistance.     

The disease resistance response in pea is associated with increased levels of specific mRNAs

A cDNA library was constructed using poly(A)⁺RNA fromPisum sativum which had been treated for 8 h with the fungusFusarium solani f. sp.phaseoli. Two thousand four hundred recombinant colonies were screened by differential colony hybridization using³²P-labelled cDNAs prepared from RNA extracted from either noninoculated or inoculated pea tissue. cDNA clones were then selected, which showed greater hybridization with cDNA prepared from pea RNA 8 h post-inoculation than with a cDNA probe from 0 h. Seven distinct hybridization classes were chosen for further study. Northern blot analyses of total cellular RNAs inoculated for 16 h with eitherF. solani phaseoli or water demonstrated that each cDNA clone selected represents an mRNA species which increases substantially in abundance during infection. Results of³H-uridine pulse-labelling experiments suggested that enhanced synthesis is at least partially responsible for the accumulation of the fungus-inducible mRNAs which hybridized with the clones.     

Ribosomal Competence and Spore Germination in Fusarium solani

Extracts prepared from macroconidia of Fusarium solani f. sp. phaseoli are capable, under defined conditions, of incorporating phenylalanine into polypeptide with exogenous polyuridylic acid as messenger. Extracts from ungerminated and germinated spores have approximately the same activity. With endogenous template, leucine incorporation occurs, but in this reaction extracts from germinated spores have about 10 times more activity than do those from ungerminated spores. It is suggested that the low rate in ungerminated spores is attributable to a relative deficiency in the number of ribosomes which are organized into polysomes.     

Acquired resistance of Alternaria solani and Sclerotium rolfsii to Polyoxin-D

Acquired resistance to the antibiotic polyoxin-D was studied in two phytopathogenic fungi, Alternaria solani Sorauer, and Sclerotium rolfsii Sacc. The ED₅₀ value of the antibiotic for A. solani was 100 μg/ml and S., rolfsii 200 μg/ml. A. solani and S. rolfsii could be trained to tolerate concentrations upto 1.000 μg/ml and 2.200 μg/ml respectively. The acquired resistance in both cases was not lost on continued subculturing in fungicide-free, media. On transfer to fungicide-free media, Polyoxinresistant strains of both the fungi showed faster growth rate and appreciable reduction in sporulation compared to the original strains. The adapted strain of A. solani showed cross-resistance to Cycloheximide and Difolatan but not to Hinosan and Bayleton.     

Sulphur and phosphorus requirements of three fungi causing diseases in storage

Summary The effect of different sulphur and phosphorus compounds on the growth and reproduction of three fungi causing storage rot, viz.,Fusarium solani, Botryodiplodia ananassae andMacrophomina phaseoli has been studied. Sixteen different sources of sulphur were used and out of them magnesium sulphate was found to be most favourable for the growth and reproduction of all the three fungi. Sodium sulphite and sodium bisulphite were toxic. Potassium metabisulphite prevented growth ofF. solani and M. phaseoli while it supported moderate growth ofB. ananassae. Only magnesium sulphate could induce the sporulation ofB. ananassae while sporulation and sclerotial development ofF. solani andM. phaseoli respectively varied with the type of sulphur sources used. Optimum concentration of magnesium sulphate was also determined and it was found that the growth and sporulation ofF. solani andB. ananassae were best at 0.375 g/l and 0.75 g/l.M. phaseoli tolerated higher doses of this substance as the best growth and excellent sclerotial development were recorded at 3.0 g/l (the maximum concentration used). Phosphorus was found to be essential for the present fungi as none of them could grow in complete absence of this substance. Onthophosphates and nucleic acid, were found to be favourable sources for growth and reproduction of the 3 organisms.     

An improved test for evaluating the pathogenicity of isolates of Fusarium solani f.sp. pisi on pea

An improved in vitro test is described for determining the pathogenicity of Fusarium solani f.sp. pisi isolates on pea. This technique involves the use of polypropylene fibre Milcap plugs to suspend peas in boiling tubes containing spore suspensions in 0.1% water agar. Results were available after 14 days of incubation at 25°C. Four levels of pathogenicity were detected on pea cultivars Little Marvel and Dark Skinned Perfection using a total of eight isolates and strains of F. solani f.sp. pisi.     

Characterization of a 20 kDa DNase elicitor from Fusarium solani f. sp. phaseoli and its expression at the onset of induced resistance in Pisum sativum

DNase released from Fusarium solani f. sp. phaseoli (Fsph DNase) has previously been reported to induce pathogenesis-related (PR) genes, phytoalexin accumulation and disease resistance against subsequent challenge with the true pea pathogen, Fusarium solani f. sp. pisi (Fspi). This report is a further analysis of DNase production with probes specific for both the gene and protein. N-terminal analysis of the ≈20 kDa Fsph DNase protein facilitated both the development of anti-Fsph DNase antiserum and the cloning of the Fsph DNase gene. Utilizing the anti-Fsph DNase antiserum to prepare an affinity column, we demonstrated that the retention and recovery of the DNase activity was associated with this protein. Fsph DNase protein was detectable by Western analysis in both the fungi and plant cytoplasm within 6–8 h following inoculation of the pea endocarp surface. Partially purified DNase detected via catalytic activity began accumulating within pea tissue at 3 h post-inoculation. Enhanced fragmentation of pea DNA occurred within 5 h following treatment of pods with Fsph DNase or inoculations with the two fungi. DNA cleavage within the nuclei of endocarp pea cells was detectable via a TUNEL assay at 3 h post-inoculation. As a result of these findings, we propose that the entrance of Fsph DNase into the pea cell and the signalling of plant defence responses is temporally associated with the damage of host DNA.     

Genetic homogeneity among isolates of Fusarium solani that cause soybean sudden death syndrome

  Fusarium solani f. sp. phaseoli is the etiological agent of soybean sudden death syndrome (SDS). This form species includes both members that cause SDS and those that do not. Despite the extensive use of SDS isolates in soybean plant breeding studies, no information regarding genetic relatedness of isolates is available. Sequencing of the D2 region of the large-subunit (28S) ribosomal DNA of 19 isolates of F. solani f. sp. phaseoli, both SDS and non-SDS isolates, resulted in identical sequences and thus indicated a very low level of genetic variation within the form species. Sequencing of the ITS region resulted in low-level intra-individual as well as intra-specific variation. Random amplified polymorphic DNA (RAPD) analysis was used for a genome-wide estimate of genetic variation and was able to resolve only two amplitypes of the SDS isolates. Thus, SDS isolates from throughout the U.S. comprise an almost clonal population with an extremely low level of genetic variation among individuals. Received: 22 November 1996 / Accepted: 4 April 1997     

Pea genes associated with non-host disease resistance to Fusarium are also active in race-specific disease resistance to Pseudomonas

A given plant species is able to resist most of the potentially pathogenic microorganisms with which it comes in contact. This phenomenon, known as non-host resistance, can be overcome only by a very small number of true pathogens which can use that plant as a host. In some cases, plants have developed mechanisms for overcoming infection by specific races or strains of a true pathogen. This race-specific resistance can be easily manipulated into agronomically important cultivars by plant breeders. We have previously described nine cDNA clones which represent pea genes active during non-host resistance against the fungus Fusarium solani f. sp. phaseoli. In the present work, we have used these cDNAs as probes to compare non-host resistance with race-specific responses of peas against three races of Pseudomonas syringae pv. pisi. Five of the genes most active during non-host resistance were also active in direct correlation with the phenotypic expression of resistance in race-specific reactions of five differential pea cultivars against three races of Pseudomonas syringae pv. pisi.     

Antimicrobial activity of Araucaria cunninghamii Sweet and the chemical constituents of its twigs and leaves

The present study was aimed at the identification of antimicrobial components from Araucaria cunninghamii with an activity-guided purification process. Eight compounds were obtained from the most active n-BuOH fraction and identified as the new compound 4-n-butoxyl-phenylpropanetriol (1), together with seven known compounds (2–8). These compounds were tested for antimicrobial activities against five bacteria and four plant pathogenic fungi. Within the series of compounds tested, compound 2 was the most active, particularly displaying moderate antibacterial activities against Erwinia carotovora and Bacillus subtilis with MICs 7.8 and 15.5 μg/ml. Moreover, this compound exhibited inhibitory activities against four plant pathogenic fungi: Helminthosporium sativum, Rhizoctonia solani, Fusarium oxysporum f. sp. Niveum and Fusarium oxysporum f. sp. Cubense, with EC₅₀ values of 42.3, 90.0, 62.7 and 100.2 μg/ml. To our knowledge, this is the first report that the n-BuOH fraction and compound 2 from A. cunninghamii showed inhibitory activity against plant pathogenic fungi.     

Genetic Diversity of Fusarium oxysporum Strains from Common Bean Fields in Spain

Fusarium wilt is an endemic disease in El Barco de Avila (Castilla y León, west-central Spain), where high-quality common bean cultivars have been cultured for the last century. We used intergenic spacer (IGS) region polymorphism of ribosomal DNA, electrophoretic karyotype patterns, and vegetative compatibility and pathogenicity analyses to assess the genetic diversity within Fusarium oxysporum isolates recovered from common bean plants growing in fields around El Barco de Avila. Ninety-six vegetative compatibility groups (VCGs) were found among 128 isolates analyzed; most of these VCGs contained only a single isolate. The strains belonging to pathogenic VCGs and the most abundant nonpathogenic VCGs were further examined for polymorphisms in the IGS region and electrophoretic karyotype patterns. Isolates belonging to the same VCG exhibited the same IGS haplotype and very similar electrophoretic karyotype patterns. These findings are consistent with the hypothesis that VCGs represent clonal lineages that rarely, if ever, reproduce sexually. The F. oxysporum f. sp. phaseoli strains recovered had the same IGS haplotype and similar electrophoretic karyotype patterns, different from those found for F. oxysporum f. sp. phaseoli from the Americas, and were assigned to three new VCGs (VCGs 0166, 0167, and 0168). Based on our results, we do not consider the strains belonging to F. oxysporum f. sp. phaseoli to be a monophyletic group within F. oxysporum, as there is no correlation between pathogenicity and VCG, IGS restriction fragment length polymorphism, or electrophoretic karyotype.