Induction of defense responses in cultured parsley cells by plant cell wall fragments

Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased β-1,3-glucanase activity when treated with either a purified α-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l-methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense.     

Stress Responses in Alfalfa (Medicago sativa L.): I. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures

Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes l-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone O-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonia-lyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid O-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-β-d-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression.     

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.     

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.     

AM真菌和胞囊线虫对大豆根内酶活性的影响*

将‘鲁豆4号’大豆接种丛枝菌根(AM)真菌聚生球囊霉Glomus fasiculatum和大豆胞囊线虫(SCN)Heterodera glycines 4号生理小种后, 定期测定大豆根系中AM真菌及线虫侵染速率、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、β-1,3葡聚糖酶及几丁质酶活性的动态变化。结果表明, 接种AM真菌大豆根系中4种酶活性高于对照水平; 先接种AM真菌后接种SCN处理根系中POD、PAL及几丁质酶的活性高于只接种SCN的处理,并且酶活性峰值出现的时间均早于或相当于后者。另外,PAL及几丁质酶活性出现高峰时期也正是AM真菌侵染率迅速升高及线虫侵染速率快速下降期。因此,AM真菌先激活了大豆的防御反应,然后使其对SCN的侵染产生快速反应,PAL及几丁质酶在AM真菌诱导的抗、耐线虫病害机制中起重要作用。值得注意的是,先接种AM真菌后接种SCN处理大豆根系中,β-1,3葡聚糖酶活性低于只接种AM真菌的处理。作者认为本试验条件下,该酶在大豆抗SCN病害中的作用表现不明显。     

Identification of Several Pathogenesis-Related Proteins in Tomato Leaves Inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-beta-Glucanases and Chitinases

Inoculation of tomato (Lycopersicon esculentum) leaves with Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) results in a marked accumulation of several pathogenesis-related (PR) proteins in the apoplast. Two predominant PR proteins were purified from apoplastic fluid by ion exchange chromatography followed by chromatofocusing. One protein (molecular mass [M_r] 35 kilodaltons [kD], isoelectric point [pI] ~6.4) showed 1,3-β-glucanase activity, while the other one (M_r26 kD, pI ~6.1) showed chitinase activity. Identification of the products that were released upon incubation of the purified enzymes with laminarin or regenerated chitin revealed that both enzymes showed endo-activity. Using antisera raised against these purified enzymes from tomato and against chitinases and 1,3-β-glucanases isolated from other plant species, one additional 1,3-β-glucanase (M_r33 kD) and three additional chitinases (M_r 27, 30, and 32 kD) could be detected in apoplastic fluids or homogenates of tomato leaves inoculated with C. fulvum. Upon inoculation with C. fulvum, chitinase and 1,3-β-glucanase activity in apoplastic fluids increased more rapidly in incompatible interactions than in compatible ones. The role of these hydrolytic enzymes, potentially capable of degrading hyphal walls of C. fulvum, is discussed in relation to active plant defense.     

Role of chitinase and beta-1,3-glucanase activities produced by a fluorescent pseudomonad and in vitro inhibition of Phytophthora capsici and Rhizoctonia solani

A study was conducted to investigate the possibility of involvement of chitinase and beta-1,3-glucanase of an antagonistic fluorescent Pseudomonas in growth suppression of phytopathogenic fungi, Phytophthora capsici and Rhizoctonia solani. Fluorescent Pseudomonas isolates GRC(3) and GRC(4) were screened for their antifungal potential against phytopathogenic fungi by using dual culture technique both on solid and liquid media. The percent inhibition was calculated. Various parameters were monitored for optimization of enzyme activities by fluorescent Pseudomonas GRC(3). The involvement of chitinases, beta-1,3-glucanases, and antifungal metabolites of nonenzymatic nature was correlated with the inhibition of P. capsici and R. solani. The results provide evidence for antibiosis as a mechanism for antagonism. The study also confirms that multiple mechanisms are involved in suppressing phytopathogens as evidenced by the involvement of chitinase and beta-1,3-glucanase in inhibition of R. solani but not P. capsici by isolate GRC3.     

Isolation and Characterization of Trichoderma spp. for Antagonistic Activity Against Root Rot and Foliar Pathogens

Trichoderma, soil-borne filamentous fungi, are capable of parasitising several plant pathogenic fungi. Twelve isolates of Trichoderma spp. isolated from different locations of South Andaman were characterized for their cultural, morphological and antagonistic activity against soil borne and foliar borne pathogens. The sequencing of these isolates showed seven different species. The isolates revealed differential reaction patterns against the test pathogens viz., Sclerotium rolfsii, Colletotrichum gloeosporioides and C. capsici. However, the isolates, TND1, TWN1, TWC1, TGD1 and TSD1 were most effective in percentage inhibition of mycelial growth of test pathogens. Significant chitinase and β-1,3-glucanase activities of all Trichoderma isolates has been recorded in growth medium. T. viride was found with highest chitinase whereas T. harzianum was recorded with highest β-1,3-glucanase activities.     

Acibenzolar-S-methyl induced resistance to <Emphasis Type="Italic">Phytophthora capsici</Emphasis> in pepper leaves

The leaves of pepper (Capsicum anuum L.) were inoculated with Phytophthora capsici Leonian 3 d after treatment with acibenzolar-S-methylbenzo [1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester (ASM) and resistance to Phytophthora blight disease was investigated. Results showed that P. capsici was significantly inhibited by ASM treatment by up to 45 % in planta. The pepper plants responded to ASM treatments by rapid and transient induction of L-phenylalanine ammonia-lyase (PAL), increase in total phenol content and activities of chitinase and β-1,3-glucanase. No significant increases in enzyme activities were observed in water-treated control plants compared with the ASM-treated plants. Therefore it may be suggested that ASM induces defense-related enzymes, PAL activity, PR proteins and phenol accumulation in ASM-treated plants and contribute to enhance resistance against P. capsici.     

The study of mycolytic properties of aerobic spore-forming bacteria producing extracellular chitinases

The mycolytic activity of 27 strains of antagonistic bacilli belonging to two taxonomic groups (18 strains of Bacillus subtilis and 9 strains of Paenibacillus ehimensis) capable of induced synthesis of chitinolytic enzymes was studied. Most of the B. subtilis strains neither displayed visible mycolytic effects on the phytopathogenic fungus Bipolaris sorokiniana in vitro, nor produced chitinases in the presence of an autoclaved mycelium. On the contrary, P. ehimensis strains grown under conditions favorable for induction of chitinases and other hydrolases exhibited a pronounced lytic effect on B. sorokiniana and actively grew by utilizing mycelium as the sole source of carbon and nitrogen. Comparison of the mycolytic activities of extracellular hydrolases in the studied strains demonstrated low correlation between chitinase production and the ability of the strains to degrade the cell walls of B. sorokiniana. Characterization of enzyme profiles in the studied strains revealed that β-1,3-glucanase was a more significant factor than chitinase for determining the mycolytic potential of bacteria and their ability to utilize the mycelium of phytopathogenic fungi as a growth substrate.     

Induction of defence-related enzymes in susceptible variety of banana: role of Fusarium-derived elicitors

Elicitor prepared from the Fusarium oxysporum f. sp cubense (Foc) isolated from infected banana rhizosphere induced the accumulation of resistance-associated enzymes in leaves of susceptible and resistant variety of banana. Roots of Grand Naine (susceptible) and robusta (resistant) variety were inoculated with 1 g/l Foc elicitors. Distinct difference in peroxidase, polyphenol oxidase, β-1,3-glucanase, chitinase and phenolics was observed in control plants of resistant and susceptible varieties. Induced defence-related enzymes in susceptible variety were increased tothe level of untreated resistant variety. This depicted that Fusarium-derived elicitor effectively induced defence in susceptible variety to the apparent level of untreated resistant variety.     

Elicitation of Diterpene Biosynthesis in Rice (Oryza sativa L.) by Chitin

Cell-free extracts of UV-irradiated rice (Oryza sativa L.) leaves have a much greater capacity for the synthesis from geranylgeranyl pyrophosphate of diterpene hydrocarbons, including the putative precursors of rice phytoalexins, than extracts of unstressed leaves (KA Wickham, CA West [1992] Arch Biochem Biophys 293: 320-332). An elicitor bioassay was developed on the basis of these observations in which 6-day-old rice cell suspension cultures were incubated for 40 hours with the substance to be tested, and an enzyme extract of the treated cells was assayed for its diterpene hydrocarbon synthesis activity as a measure of the response to elicitor. Four types of cell wall polysaccharides and oligosaccharide fragments that have elicitor activity for other plants were tested. Of these, polymeric chitin was the most active; a suspension concentration of approximately 7 micrograms per milliliter gave 50% of the maximum response in the bioassay. Chitosan and a branched β-1,3-glucan fraction from Phytophthora megasperma f. sp. glycinea cell walls were only weakly active, and a mixture of oligogalacturonides was only slightly active. A crude mycelial cell wall preparation from the rice pathogen, Fusarium moniliforme, gave a response comparable to that of chitin, and this activity was sensitive to predigestion of the cell wall material with chitinase before the elicitor assay. N-Acetylglucosamine, chitobiose, chitotriose, and chitotetrose were inactive as elicitors, whereas a mixture of chitin fragments solubilized from insoluble chitin by partial acid hydrolysis was highly active. Constitutive chitinase activity was detected in the culture filtrate and enzyme extract of cells from a 6-day-old rice cell culture; the amount of chitinase activity increased markedly in both the culture filtrate and cell extracts after treatment of the culture with chitin. We propose on the basis of these results that soluble chitin fragments released from fungal cell walls through the action of constitutive rice chitinases serve as biotic elicitors of defense-related responses in rice.     

Chitinolytic activity of Bacillus Cohn.--phytopathogenic fungus antagonist

Among the 70 tested Bacillus spp. strains antagonistic to phytopathogenic fungi, 19 were found to possess chitinolytic activity when grown on solid media with 0.5% colloidal chitin. The chitinolytic activity of almost all of these 19 strains grown in liquid cultures ranged from 0.1 to 0.3 U/ml. One of the 19 strains exhibited exochitinase activity. In addition to chitinase, two strains also produced chitosanase and one strain, beta-1,3-glucanase. No correlation was found between the antifungal activity of the bacillar strains studied and their ability to synthesize extracellular chitinase. Among the 19 chitinolytic strains, the correlation between these parameters was also low (r x,y = 0.45), although the enzymatic preparations of most of these strains inhibited the growth of the phytopathogenic fungus Helminthosporium sativum.     

Biochemical Plant Responses to Ozone : III. Activation of the Defense-Related Proteins beta-1,3-Glucanase and Chitinase in Tobacco Leaves

A single pulse of O₃ (0.15 microliter per liter, 5 hours) induced β-1,3-glucanase and chitinase activities in O₃-sensitive and -tolerant tobacco (Nicotiana tabacum L.) cultivars. In the O₃-sensitive cultivar Bel W3, the response was rapid (maximum after 5 to 10 hours) and was far more pronounced for β-1,3-glucanase (40- to 75-fold) than for chitinase (4-fold). In the O₃-tolerant cultivar Bel B, β-1,3-glucanase was induced up to 30-fold and chitinase up to 3-fold under O₃ concentrations that did not lead to visible damage. Northern blot hybridization showed a marked increase in β-1,3-glucanase mRNA in cultivar Bel W3 between 3 and 24 hours following O₃ treatment, a transient induction in cultivar Bel B, and no change in control plants. The induction of β-1,3-glucanase and chitinase activities following O₃ treatment occurred within the leaf cells and was not found in the intercellular wash fluids. In addition, O₃ treatment increased the amount of the β-1,3-glucan callose, which accumulated predominantly around the necrotic spots in cultivar Bel W3. The results demonstrate that near-ambient O₃ levels can induce pathogenesis-related proteins and may thereby alter the disposition of plants toward pathogen attack.     

Tobacco Mesophyll Protoplasts Synthesize 1,3-beta-Glucanase, Chitinases, and "Osmotins" during in Vitro Culture

Tobacco (Nicotiana tabacum) mesophyll protoplasts synthesize six basic proteins (a, a′, a₁, b, b′, and c) which are undetectable in the leaf and whose synthesis is reduced by auxin (Y Meyer, L Aspart, Y Chartier [1984] Plant Physiol 75: 1027-1033). Polypeptides a, a′, and a₁ were shown to have similar mobilities on two-dimensional electrophoresis as one 1,3-β-glucanase and two chitinases from tobacco mosaic virus-infected leaves. In immunoblotting experiments, polypeptide a was recognized by specific antibodies raised against the 1,3-β-glucanase and a′ and a₁ reacted with anti-chitinase antibodies. Similarly, b and b′ comigrated with osmotin and its neutral counterpart, two proteins characteristic of salt-adapted tobacco cells, and reacted with anti-osmotin antibodies. In addition it has been shown that 1,3-β-glucanase and chitinase activities increased at the same time as a, a′, and a₁ accumulated in cultivated protoplasts. Finally, polypeptide c was also detected in tobacco mosaic virus-infected leaves but could not be identified as any of the pathogenesis-related proteins characterized so far in tobacco. Thus, cultivated tobacco protoplasts synthesize and accumulate typical stress proteins.     

Characterization of a chitinase and an endo-beta-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Of 24 Trichoderma isolates, T harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and beta-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa beta-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (beta-1,3-glucanase). Km and Kcat were 3.8 g l(-1) and 0.71 s(-1) for the chitinase (chitin) and 1.1 g(-1) and 52 s(-1) for the beta-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the beta-1,3-glucanase was specific for beta-1,3-linkages in polysaccharides. Both enzymes were stable at 30 degrees C, while at 60 degrees C the chitinase and the beta-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 microg/ml.     

Biological function of ;pathogenesis-related' proteins: four PR proteins of tobacco have 1,3-beta-glucanase activity

Three of the ten acidic `pathogenesis-related' (PR) proteins known to accumulate in Nicotiana tabacum cv Samsun NN reacting hypersensitively to tobacco mosaic virus, namely −O, −N and −2, have been shown to have 1,3-β-glucanase (EC 3.2.1.39) activity. By using sera raised against each protein purified to homogeneity close serological relationships have been demonstrated between the three proteins. The same specific sera cross-reacted with a basic protein which is also a 1,3-β-glucanase induced by virus infection and which can be considered as a new basic pathogenesis-related protein of tobacco. Protein PR-O and the basic 1,3-β-glucanase display about the same specific enzymatic activity, i.e. 50-fold and 250-fold higher than specific activities of proteins PR-N and -2 respectively.     

A study of some biochemical and histopathological responses of wet-stored recalcitrant seeds of Avicennia marina infected by Fusarium moniliforme

Although fungi cause a recognized problem during storage of recalcitrant seeds of many tropical species, there are no data to date on defence strategies of these seeds against fungal attack. To ascertain whether recalcitrant seeds of Avicennia marina elaborate compounds that might suppress fungal proliferation during hydrated storage, the production and efficacy of beta-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) were studied in relation to histopathological changes. Freshly harvested seeds had low beta-1,3-glucanase and chitinase activities and fluorescence microscopy revealed progressive deterioration of the internal tissues of these seeds associated with fungal infection during hydrated storage. In seeds treated to minimize associated fungi (clean seeds), beta-1,3-glucanase and chitinase activities increased significantly during 10 d of hydrated storage. Similar high levels of activity were observed when these seeds were experimentally infected with Fusarium moniliforme and subjected to further storage. The histopathological observations indicated delayed disease development in the 10-d clean-storage period, although the hypersensitive response was not observed. The results suggest that, although the recalcitrant seeds of A. marina elaborate some antifungal enzymes, there is a lack of effective defence strategies that might lead to successful responses against fungal infections.