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.     

Pea saponins in the pea-Fusarium solani interaction

Saponin-like compounds isolated fromPisum sativum were tested for antifungal activity, effect on pea tissue, and effect on chitin and chitosan synthesis inFusarium solani. Growth ofFusarium solani f. sp.phaseoli and f. sp.pisi macroconidia was inhibited by saponins at concentrations of 150 and 300 μg/ml, respectively. Pod endocarp tissue treated with saponins showed temporary reduction in cell viability (esterase activity); however, there was no significant reduction in resistance toF. solani f. sp.phaseoli, normally incompatible on peas. Macroconidia germinated in the presence of saponin showed decreased incorporation ofN-[³H]acetylglucosamine into chitin and chitosan at concentrations as low as 32 μg/ml. Thus, a reduction in chitin and chitosan synthesis may be associated with inhibition of fungal growth. Saponins may contribute to the disease resistance of peas     

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.     

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.     

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.     

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.     

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.     

Glycosidic Enzyme Activity in Pea Tissue and Pea-Fusarium solani Interactions

Membrane barriers which prevent direct contact between Fusarium solani and pea endocarp tissue prevent fungal spores from inducing phytoalexin production. Conversely, preinduced host resistance responses are not readily transported from the plant across the membrane barrier to Fusarium macroconidia.     

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.     

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.     

Cross-Reactive Antigens Between Pea and Some Fungal Plant Pathogens

Antiserum to pea was used to analyse cross-reactive antigens (CRA) between pea and some fungal plant pathogens with different levels of specificity towards this host by using both double diffusion and immunoblotting techniques. Non pathogens of pea were also included in the study. Nectria haematococca MPVI, the three formae speciales dianthi, lycopersici and pisi of Fusarium oxysporum and Ascochyta pisi produced strong reactions with both techniques. In N. haematococca MPI, F. solani f. sp. phaseoli, V. dahliae and Phoma medicaginis var. pinodella instead, reactions were not detected by double diffusion but only the more sensitive immunoblotting technique. No CRA were observedin, the non-specific pathogens Rhizoctonia solani, Sclerotium rolfsii and Sclerotinia sclerotiorum, as well as in the non-pathogen Phytophthora capsici. The immunoblotting patterns of the most reactive fungi showed common bands with molecular weights of 84, 75 and 62 kDa. Some bands were present only in the specific pathogens N. haematococca MPVI and F. oxysporum f.sp. pisi. The possible involvement in host-parasite interactions of cross-reactive antigens which are, present in the analyzed fungi is discussed.     

Identification of a stress-induced protein in stem exudates of soybean seedlings root-infected with Fusarium solani f. sp. glycines

Sudden death syndrome of soybean (Glycine max) is caused by the soilborne fungus, Fusarium solani f. sp. glycines, that infects soybean roots. Besides root necrosis, symptoms include interveinal leaf chlorosis, necrosis and premature defoliation. It is proposed that a fungal toxin is produced in soybean roots and translocated to foliage. In this study, we isolated compounds from soybean stem exudates from plants that were either inoculated or not inoculated with F. solani f. sp. glycines. A protein with an estimated molecular mass of 17 kDa and designated as FISP 17 for F. solani f. sp. glycines-induced stress protein was identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein occurred only in F. solani f. sp. glycines-infected soybean stem exudates. The N-terminal amino acid sequence of the purified protein had 100 % identity with a starvation-associated message 22 protein, and 80 and 78 % identity with purified bean pathogenesis-related proteins, PvPR1 and PvPR2, respectively. To determine if the protein was of plant or fungal origin, a synthetic peptide was designed based on the N-terminal sequence and used to raise a polyclonal antibody from rabbit. Western blot analysis showed that the antibody only reacted with a 17-kDa protein in F. solani f. sp. glycines-infected plant exudates, but no reaction occurred with healthy plant exudates or with culture filtrates of F. solani f. sp. glycines. This is the first report of the presence of a stress-induced protein in stem exudates of soybean seedlings root-infected with F. solani f. sp. glycines.     

12-O-tetradecanoylphorbol-13-acetate stimulates release of arachidonic acid, prostaglandin E2 and prostaglandin F2 alpha from TPA nonproliferative variants of 3T3 cells

The Proteinase Inhibitor Inducing Factor, PIIF, a pectic polysaccharide that induces synthesis and accumulation of proteinase inhibitor proteins in tomato and potato leaves, is an effective elicitor of the phytoalexin pisatin in pea pod tissues. The levels of pisatin induced by PIIF, and the time course of elicitation, are similar to those induced by chitosans, β-1,4 glucosamine polymers, which are potent elicitors of pisatin in pea pods. Similarly, the chitosans, found in both insect and fungal cell walls, are the most potent inducers yet found of proteinase inhibitor accumulation in excised tomato cotyledons. The similarity in the induction of synthesis of proteinase inhibitors in tomato cotyledons and of pisatin in pea pods by pectic polysaccharides and chitosans suggests that the two polysaccharide types may be triggering a similar fundamental system present in pea and tomato plants that regulates the expression of genes for natural protection systems.     

Proof for the Production of Cutinase by Fusarium solani f. pisi during Penetration into Its Host, Pisum sativum

Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this ferritin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection.     

The Water-Soluble Chitosan Derivative,N-Methylene Phosphonic Chitosan,Is an Effective Fungicide against the Phytopathogen Fusarium eumartii

Chitosan has been considered an environmental-friendly polymer. However, its use in agriculture has not been extended yet due to its relatively low solubility in water. N-Methylene phosphonic chitosan (NMPC) is a water-soluble derivative prepared by adding a phosphonic group to chitosan. This study demonstrates that NMPC has a fungicidal effect on the phytopathogenic fungus Fusarium solani f. sp. eumartii (F. eumartii) judged by the inhibition of F. eumartti mycelial growth and spore germination. NMPC affected fungal membrane permeability, reactive oxygen species production, and cell death. Also, this chitosan-derivative exerted antifungal effects against two other phytopathogens, Botrytis cinerea, and Phytophthora infestans. NMPC did not affect tomato cell viability at the same doses applied to these phytopathogens to exert fungicide action. In addition to water solubility, the selective biological cytotoxicity of NMPC adds value in its application as an antimicrobial agent in agriculture.     

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.     

Ultrastructural Changes Caused by Fusarium oxysporum f. sp. lycopersici in Meloidogyne javanica Induced Giant Cells in Fusarium Resistant and Susceptible Tomato Cultivars

Tomato (Lycopersicon esculentum Mill.) seedlings, susceptible (cv. Pearson A-I Improved) and resistant (cv. Pearson Improved) to race 1 Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyd &Hans., were inoculated with Meloidogyne javanica (Trueb) Chitwood second-stage juveniles and 3 weeks later with race 1 F. oxysporum f. sp. lycopersici spores. One week after fungal inoculation, no fungus was visible in root tissue of the tomato cultivars and the giant cells were normal. Two weeks after fungal inoculation, abundant hyphae were visible in xylem tissues of Fusarium-susceptible but not of Fusarium-resistant plants. In susceptible plants, giant cell degeneration occurred, characterized by membrane and organelle disruption. In addition, where hyphae were in direct contact with the giant cell, dissolution of the giant cell wall occurred. Three weeks after fungal inoculation, fungal hyphae and spores were visible inside xylem tissues and giant cells in Fusarium-susceptible plants and in xylem tissue of the resistant plants. In susceptible and resistant plants, giant cell degeneration was apparent. Giant cell walls were completely broken down in Fusarium-susceptible tomato plants. In both cultivars infected by Fusarium, giant cell nuclei became spherical and dark inclusions occurred within the chromatin material which condensed adjacent to the fragmented nuclear membrane. No such ultrastructural changes were seen in the giant cells of control plants inoculated with nematode alone. Giant cell deterioration in both cultivars is probably caused by toxic fungal metabolites.     

Molecular characterization of a pea β-1,3-glucanase induced by Fusarium solani and chitosan challenge

-glucanases are prominent proteins in pea endocarp tissue responding to fungal infection. We have cloned and sequenced a partial pea cDNA clone, pPIG312, corresponding to a -1,3-glucanase in pea pods challenged with the incompatible pathogen Fusarium solani f. sp. phaseoli. The insert from the partial pea cDNA was used to probe a genomic library derived from pea leaves of the same cultivar. One of the genomic clones, pPIG4-3, contained the complete coding sequence for a mature -1,3-glucanase protein. The predicted amino acid sequence of the pea -1,3-glucanase has 78% identity to bean -1,3-glucanase, 62% and 60% to two tobacco -1,3-glucanases, 57% to soybean -1,3-glucanase, 51% to barley -1,3-glucanase, and 48% to barley -1,3-1,4-glucanase. Genomic Southern analysis indicates that the pea genome contains only one -1,3-glucanase gene corresponding to the probe used in this study. Accumulation of -1,3-glucanase mRNA homologous with the pPIG312 probe was detected in pea pods within 4 to 8 h after challenge with F. solani f. sp. phaseoli, f. sp. pisi, a compatible strain, or the elicitor, chitosan. In the incompatible reaction, mRNA accumulation remained high for 48h, whereas it rapidly decreased in the compatible reaction. After fungal inoculation of whole pea seedlings, the enhanced mRNA accumulation occurred mainly in the basal region (lower stem and root). This -1,3-glucanase glucanase mRNA was constitutively expressed in the roots of pea seedlings. The sustained levels of -glucanase mRNA expression induced by the incompatible pathogen in the resistance response suggests that the enzyme contributes to the pea plant's general defense.     

Green synthesis approach: extraction of chitosan from fungus mycelia

Chitosan, copolymer of glucosamine and N-acetyl glucosamine is mainly derived from chitin, which is present in cell walls of crustaceans and some other microorganisms, such as fungi. Chitosan is emerging as an important biopolymer having a broad range of applications in different fields. On a commercial scale, chitosan is mainly obtained from crustacean shells rather than from the fungal sources. The methods used for extraction of chitosan are laden with many disadvantages. Alternative options of producing chitosan from fungal biomass exist, in fact with superior physico-chemical properties. Researchers around the globe are attempting to commercialize chitosan production and extraction from fungal sources. Chitosan extracted from fungal sources has the potential to completely replace crustacean-derived chitosan. In this context, the present review discusses the potential of fungal biomass resulting from various biotechnological industries or grown on negative/low cost agricultural and industrial wastes and their by-products as an inexpensive source of chitosan. Biologically derived fungal chitosan offers promising advantages over the chitosan obtained from crustacean shells with respect to different physico-chemical attributes. The different aspects of fungal chitosan extraction methods and various parameters having an effect on the yield of chitosan are discussed in detail. This review also deals with essential attributes of chitosan for high value-added applications in different fields.