Induction of Defence Responses in Roots and Mesocotyls of Sorghum Seedlings by Inoculation with Fusarium thapsinum and F. proliferatum, Wounding and Light

The defence reactions of sorghum seedlings 7 days after inoculation with Fusarium thapsinum and F. proliferatum, and interactions with wounding and exposure to light were studied to determine whether responses to these fungi differed from those to abiotic stresses. In non‐wounded plants, inoculation with both fungi increased concentrations of anthocyanins and soluble phenolics and activities of peroxidase (POX), chitinase and β‐1,3‐glucanase in the roots, and increased β‐1,3‐glucanase activity in the mesocotyls. There was no effect of inoculation on phenylalanine ammonia‐lyase (PAL) activity. Wounding by itself increased anthocyanin content of mesocotyls. Wounding also had a variety of interactions with inoculation. Exposure to light had very little effect on any defence response measured. A time course experiment showed that induction of chitinase and β‐1,3‐glucanase occurred in less than 24 h after inoculation. POX activity increased 2 days after inoculation, followed by a transient increase in PAL activity. The content of anthocyanins and soluble phenolics in roots of inoculated seedlings increased gradually compared with controls over 6 days. The responses of sorghum seedlings to inoculation with F. thapsinum and F. proliferatum were similar to those found by other workers following challenge by necrotrophic pathogens and were different from those induced by wounding and exposure to light.     

Induction of β-1,3-glucanase and chitinase activity in the defense response of Eruca sativa plants against the fungal pathogen Alternaria brassicicola

Plants have developed many mechanisms to protect themselves against most potential microbial pathogens and diseases. Pathogenesis-related proteins are produced as a part of the active defenses to prevent attack. In this study, the induction of PR proteins in Eruca sativa in response to fungal pathogen Alternaria brassicicola was investigated in 10 days and one-month-old plants. Induction of pathogen resulted in a much marked increase in the activities of β-1,3-glucanase and chitinase in resistant cultivar (RTM-2002) as compared to susceptible (T-27) one. The enzyme activity gradually increased throughout the experimental period of 168 h compare to control. However, the activation of β-1,3-glucanase and chitinase was more rapid and to a greater extent in plants of RTM-2002 than in T-27. western blot analysis revealed the presence of 33 and 32 kDa β-1,3-glucanase and chitinase in induced arugula plants, respectively. The biochemical approach described in this article with E. sativa provide the basis for further efforts concentrating on the isolation and characterization of elements involved in perception and in the early steps of intracellular signal transduction.     

Effects of Various Elicitors on the Transcription of a β-1,3-endoglucanase Gene in Citrus Fruit

Very little is yet known regarding the molecular mechanisms involved in pathogen defense responses in citrus fruit. Recently, a basic β-1,3-endoglucanase (EC 3.2.2.39) belonging to the pathogenesis-related (PR) group of proteins, has been purified from Citrus sinensis (L) Osbeck cv. `Valencia' orange callus. Specific antibodies raised against the purified protein were used to screen `Valencia' callus and flavedo cDNA expression libraries, and to isolate its corresponding cDNA, designated gns1. The gns1 gene encodes a predicted polypeptide of 336 amino acids with a molecular mass of 37.3 kDa and a basic pI of 9.19, and shares 55–65% identity with several other plant β-1,3-endoglucanase proteins. Hereby, we show that the expression of the gns1 gene is markedly induced by wounding and inoculation with Penicillium digitatum (Pers. Fr.) Sacc., and following treatments with various elicitors that induce fruit resistance against P. digitatum . These treatments include UV irradiation, application of jasmonic acid (JA), β-aminobutyric acid (BABA), Candida oleophila antagonist yeast cells and hot water rinsing and brushing. Overall, based on various RNA gel blot hybridizations, we assume that gns1 is most likely to be part of the molecular mechanisms involved in pathogen defense responses in citrus fruit. *     

Citrus rootstock responses to herbivory by larvae of the sugarcane rootstock borer weevil (Diaprepes abbreviatus)

The responses of roots to feeding by larvae of a citrus root weevil (Diaprepes abbreviatus) were investigated in Citrus grandis (L.) Osb. x Poncirus trifoliata (2N) (L.) Raf.; C. grandis x P. trifoliata (4N); P. trifoliata x C. grandis (Flying Dragon x Nakon); C. paradisi Macf. x P. trifoliata (Swingle citrumelo); C. aurantifolia (Christm.) Swingle (Citrus macrophylla); C. reticulata Blanco (Cleopatra mandarin); C. sinensis (L.) Osb. x P. trifoliata (Carrizo citrange); C. aurantium (L.) (sour orange). Chitinase, chitosanase. β-1,3-glucanase, peroxidase and lysozyme activities were measured and significant differences were observed for some of the cultivars between infested and uninfested rootstocks. Generally, increased activities were observed for chitinases and decreased activities were observed for the other enzymes measured. Numerous significant differences in hydrolase and peroxidase activities were observed between cultivars. Immunological detection revealed that new protein bands occurred in root protein extracts for six of the eight cultivars infested with larvae when an antibody to a class I potato leaf chitinase was used. Antibodies generated against two citrus chitinases of M_r 24 000 (basic chitinase cv. Valencia (C. sinensis) callus, BCVC) and M_r 28 000 (basic chitinase/lysozyme cv. Valencia callus, BCLVC) indicated that chitinases in Carrizo were induced in infested roots when the BCVC antibody was employed. These findings justify calling these proteins pathogenesis-related proteins. The chitinase that BCLVC was prepared from exhibited high lysozyme activities, and the results of western blots showed the presence of proteins at M_r 24 000 and 27 000 which are presumed to be lysozymes. Similar tests using antibodies against β-1, 3-glucanases and peroxidases indicated a diminution of protein bands that cross-reacted with infested root protein extracts compared with what occurred in controls. All of the root extracts were tested against chitosans with various percentages of acetylation; activities were linearly dependent on the amount of chitosan acetylation; i.e. the larger the amount of acetylation, the greater the activity. Significant differences in hydrolase activities were observed between infested and uninfested roots for the rootstocks using the variously acetylated substrates. All of the root protein extracts were capable of degrading peritrophic membranes removed from larvae of D. abbreviatus. This suggests that citrus chitinases may play a role in disrupting the peritrophic membrane such that ingested substances that pose a hazard to the insect may penetrate the membrane more easily.     

Induction and Impact of Cell Wall Degrading Enzymes in Nutrient Solution of Closed Hydroponic Systems

The potential of the microflora in nutrient solutions to produce cell wall degrading enzymes (CWDE) was investigated by adding glucose or substrates of CWDE, such as chitin, cellulose, curdlan and preparations of fungal mycelia (0, 0.01 and 0.1%, w/v). The results indicate the potential of the microflora in nutrient solutions to produce proteolytic, chitinolytic, cellulolytic as well as β‐1,3‐glucanolytic enzymes. All enzyme complexes were induced by addition of preparations of Fusarium oxysporum f. sp. cyclaminis (Focy) and Pythium ultimum, respectively. In contrast, addition of glucose to nutrient solution resulted in only slight increase of protease and chitinase. No correlation between increased activity of CWDE and survival of Focy was found.     

Induction of chitinase and β-1,3-glucanase in Parthenocissus quinquefolia cells cultured in vitro

Chitin, chitosan and peptidoglycan induced chitinase (EC 3. 2. 1. 14) activity in Parthenocissus quinquefolia cells cultured in vitro, while cellulose did not. The real inducers seemed to be oligomers released from the large size polymers by hydrolytic enzymes secreted into the medium during the cell growth and division. This effect was mimicked by the addition to the medium of a partially purified Parthenocissus chitinase/lysozyme (EC 3. 2. 1. 17), which was also able to hydrolyse chitosan. Oligomers of chitin and of chitosan induced the activity to the same level and with the same time course, while peptidoglycan oligomers induced less activity. Oligomers also induced β-1,3-glucanase (EC 3. 2. 1. 6) activities. The changes with time of both activities and the relative effects of the three kinds of polymers suggested that the induction of both enzymes involves a common element early in the signal pathway.     

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.     

Ultrastructural and Immunocytochemical Studies on Effects of Barley Yellow Dwarf Virus – Infection on Fusarium Head Blight, Caused by Fusarium graminearum, in Wheat Plants

The interactions between barley yellow dwarf virus (BYDV) and Fusarium head blight (FHB), caused by Fusarium graminearum, were studied in the two winter wheat cultivars (cvs.), Agent (susceptible to FHB) and Petrus (moderately resistant to FHB), using ultrastructural and immunocytochemical methods. Infections of wheat plants of both cvs. by BYDV increased susceptibility to FHB. BYDV infection caused numerous cytological changes in lemma tissue of both cvs. such as formation of vesicles in the cytoplasm, degradation of fine structures of chloroplasts of both cvs. and accumulation of large starch grains in the chloroplasts. Electron microscopical studies showed that the development of F. graminearum on spike surfaces was not affected in BYDV‐infected plants. After penetration and intercellular growth in lemma tissue, defence responses to Fusarium infections were markedly reduced in BYDV‐diseased plants compared to the tissue of virus‐free plants. At sites of contact of fungal cells with host tissue, depositions of cell wall material were distinctly less pronounced than in tissues of virus‐free plants of cv. Petrus. Detection of β‐1,3‐glucanases and chitinases in lemma tissue of cv. Agent revealed no appreciably increased accumulation of both defence enzymes in F. graminearum‐infected virus‐free and BYDV‐infected tissues compared to the non‐infected control tissue. On the other hand, in cv. Petrus, infection with F. graminearum induced a markedly enhanced activity of both enzymes 3 days after inoculation. The increase of both enzyme activities was less pronounced in BYDV‐infected plants than in tissue exclusively infected with F. graminearum. Cytological studies suggest that in contrast to the susceptible cv. Agent postinfectional defence responses may play still an important role in the resistance of the moderately resistant cv. Petrus to FHB.     

Induction of Enzymes and Phenolic Compounds Related to the Natural Defence Response of Netted Melon Fruit by a Bio-elicitor

Fungal disease in netted melon fruit is an important factor affecting their postharvest quality and therefore an important cause of large economic losses around the world. Among the alternatives to control fungal diseases, the induction of the natural defence response (NDR) in fruits is promising. The objective of this study was to induce the NDR in netted melon treated with a bio-elicitor formulated from Fusarium oxysporum growth in a potato dextrose agar enriched with netted melon skin. Netted melon fruits (cv 'Primo') were not treated (C), untreated and inoculated with F. oxysporum (IN), treated with a bio-elicitor (FES), or treated with the bio-elicitor and inoculated (FES + IN). After treatments, fruits were stored for 8 days at 20°C with 90–92% relative humidity. Melon was sampled every 2 days at 20°C to evaluate the development of Fusarium rot symptoms as disease index percentage (DI), changes in phenolic compounds, changes in phenylalanine ammonia-lyase (PAL) activity, chitinase activity (ChA) and β-1,3-glucanase activity (GA). It was found that DI in netted melon fruit was significantly reduced in the FES + IN as compared with the IN treatment. FES + IN and FES treatments showed the highest increase of phenolic acids. Higher levels of PAL activity were observed in the treatments IN, FES, and FES + IN with respect to C, after 4 days of storage. A large increase in ChA activity was observed in the treatments IN, FES and FES + IN after 6 days of storage. No differences in GA activity were found among FES, FES + IN and C treatments throughout storage. IN treatment showed the highest increase in GA activity after 4 days of storage. It is concluded that the bio-elicitor activates the NDR as measured by the increase in phenolic acids synthesis, PAL and ChA enzymes activity, in a similar way as the infection by the living pathogen.     

Immunohistological Analysis of Chemically Induced Proteins in Sugar Beet

Tissue-specific distribution of basic β-1,3-glucanase (Glu2), basic class II chitinase (Ch2), basic class IV chitinase (Ch4), and acidic class III chitinase (SE2) were examined both in leaves and roots of sugar beet treated with salicylic acid (SA), benzothiadiazole (BTH) and glycine betaine. Protein localization was monitored by immunohistological analysis using specific antibodies. BTH, SA as well as glycine betaine induced both Glu2 and chitinase isozymes in leaves and roots of treated plants. The enzymes were accumulated in extracellular space and cell walls. They were mostly deposited in parenchyma cells of leaves and cortex parenchyma and endodermis of roots. In leaf tissues, BTH and SA induced proteins more effectively than glycine betaine but the effect of glycine betaine in roots was as efficient as BTH and SA. Glycine betaine induced the formation of extracellular globuli containing Ch4. Induced proteins were spatially distributed over the whole plant regardless the site of the inducer application. This revised version was published online in July 2006 with corrections to the Cover Date.     

Control of Late Blight (Phytophthora capsici ) in Pepper Plant with a Compost Containing Multitude of Chitinase-producing Bacteria

Compost sustaining a multitude of chitinase-producing bacteria was evaluated in a greenhouse study as a soil amendment for the control of late blight (Phytophthora capsici L.) in pepper (Capsicum annuum L.). Microbial population and exogenous enzyme activity were measured in the rhizosphere and correlated to the growth and health of pepper plant. Rice straw was composted with and without a chitin source, after having been inoculated with an aliquot of coastal area soil containing a known titer of chitinase-producing bacteria. P. capsici inoculated plants cultivated in chitin compost-amended soil exhibited significantly higher root and shoot weights and lower root mortality than plants grown in pathogen-inoculated control compost. Chitinase and β-1,3-glucanase activities in rhizosphere of plants grown in chitin compost-amended soil were twice that seen in soil amended with control compost. Colony forming units of chitinase-producing bacteria isolated from rhizosphere of plants grown in chitin compost-amended soil were 10³ times as prevalent as bacteria in control compost. These results indicate that increasing the population of chitinase-producing bacteria and soil enzyme activities in rhizosphere by compost amendment could alleviate pathogenic effects of P. capsici.     

Extracellular hydrolases of strain <Emphasis Type="Italic">Bacillus</Emphasis> sp. 739 and their involvement in the lysis of micromycete cell walls

The mycolytic bacterial strain Bacillus sp. 739 produces extracellular enzymes which degrade in vitro the cell walls of a number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process correlated with the levels of the β-1,3-glucanase and protease activity. The comparative characteristic of Bacillus sp. 739 enzymatic preparations showed that efficient hydrolysis of the fungus cell walls was the result of the action of the complex of enzymes produced by the strain when grown on chitin-containing media. Among the enzymes of this complex, chitinases and β-1,3-glucanases hydrolyzed most actively the disintegrated cell walls of B. sorokiniana. However, only β-1,3-glucanases were able to degrade the cell walls of native fungal mycelium in the absence of other hydrolases, which is indicative of their key role in the mycolytic activity of Bacillus sp. 739.     

Induction of early blight resistance in tomato by <Emphasis Type="Italic">Quercus infectoria</Emphasis> gall extract in association with accumulation of phenolics and defense-related enzymes

Treatment of tomato leaves with aqueous extract (0.5%) of the galls of Quercus infectoria significantly reduced infection from subsequent inoculation with Alternaria solani, the tomato early blight pathogen. When the leaves were challenge-inoculated with A. solani 3 d after application of Q. infectoria gall extract (QIGE), the percent defoliation decreased from 33.6 to 7.3. Two to three day pre-treatment with QIGE reduced the percent defoliation by 77 percent. The biochemical responses of tomato plants to QIGE were also studied. In tomato plants treated with QIGE, phenolic content increased rapidly, reached the maximum at 2 d after treatment. Phenylalanine ammonia-lyase (PAL) activity increased significantly from 1 d after treatment and the maximum enzyme activity was recorded 2 d after treatment at which period a 3-fold increase in PAL activity was observed when compared to the control. Peroxidase (PO) activity was also significantly increased 1 d after treatment and the maximum activity was reached 2 d after treatment. Peroxidase isozyme analysis indicated that PO-1 was increased dramatically in tomato leaves 1 d after treatment and maintained at the same level throughout the experimental period of 6 d. When tomato leaves were treated with QIGE, a two-fold increase in chitinase and β-1,3-glucanase activities was recorded 2 and 3 d respectively, after treatment. The enhanced activities of defense-related enzymes and elevated levels of phenolics in QIGE-treated tomato plants between 1 and 3 d after treatment suggest that these induced biochemical defenses may be involved in the suppression of early blight by QIGE.     

Differential induction of acquired resistance and PR gene expression in tobacco by virus infection,ethephon treatment,UV light and wounding

Genes for acidic, extracellular and basic, intracellular pathogenesis-related (PR) proteins of tobacco were studied for their response to tobacco mosaic virus (TMV) infection, ethephon treatment, wounding and UV light. The genes encoding the acidic PR proteins (PR-1, PR-2, PR-3, PR-4 and PR-5) responded similarly to the different forms of stress. They appeared to be highly inducible by TMV, moderately inducible by ethephon treatment and UV light and not inducible by wounding. The genes for the basic counterparts of PR-1, PR-2, PR-3 and PR-5 also displayed a common stress response. However, this response was different from that of the acidic PR proteins. Here, the highest induction was obtained upon ethephon treatment, while the other stress conditions resulted in somewhat lower levels of expression. Most genes for acidic PR proteins are systemically induced in the uninfected upper leaves of TMV-infected plants, whereas the genes encoding the basic PR proteins are not. Increased levels of resistance to TMV, comparable to resistance obtained by pre-infection with the virus, were found in UV-irradiated leaves but not in wounded or ethephon-treated leaves. This indicates that the basic PR proteins are not involved in resistance to TMV infection. Tobacco phenylalanine ammonia-lyase genes were not inducible by the various stress conditions. The implications of these findings in relation to the phenomenon of acquired resistance are discussed.     

Differential accumulation of mRNAs encoding extracellular and intracellular PR proteins in tomato induced by virulent and avirulent races of Cladosporium fulvum

Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a -1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa -1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic -1,3-glucanase and a basic 35 kDa -1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic -1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa -1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.     

Effects of fungal infection and wounding on the expression of chitinases and β-1,3 glucanases in near-isogenic lines of barley

Chitinases (EC 3.2.1.14) and β -1.3 glucanases (EC 3.2.1.39) have been known to play a vital role in the defense of plants against fungal pathogens. The pattern of induction of these two enzymes subsequent to infection by powdery mildew was studied in 10 pairs of near-isogenic lines of barley ( Hordeum vulgare L.) which possess powdery mildew resistance genes. These isogenic lines have been grotiped according to their reaction to the fungus. The induction patterns varied between the resistant and the susceptible cultivars within each group and between different groups. More tsozymcs were induced in susceptible varieties of highly resistant groups and the overall levels and the number of isozymes of chitinases and β -1.3 glucanases were lower in groups with low resistance. The effect of powdery mildew infection and mechanical wounding on the cellular localization of chitinases and β -1.3 glucanases in barley leaves has also been studied. The 31 kDa leaf chitinase, L-CH2, and trace amounts of a 25 kDa chitinase. L-CH3. were present in healthy leaves. Wounding increased the levels of L-CH3 within I ft h. Powdery mildew infection increased the levels of L-CH3 both in intercellular fluid and in intracellular extract of leaves. A /3-I.3 glucanase. GH, also increased after infection and wounding. In infected barley leaves, GL-1 was present both in intercellular space and intracellular extract. It is concluded that powdery mildew resistance genes exhibit qualitative and quantitative differences in the expression of chitinases and β -1.3 glucanases. Further, chitinases and β -1.3 glucanases appear to be a response to active infection rather than the factors responsible for disease resistance.     

Altered expression of an ankyrin-repeat protein results in leaf abnormalities,necrotic lesions,and the elaboration of a systemic signal

Summary The PR-like proteins, class I -1,3-glucanase (GLU I) and chitinase (CHN I), are induced as part of a stereotypic response that can provide protection against viral, bacterial, and fungal pathogens. We have identified two Nicotiana plumbaginifolia ankyrin-repeat proteins, designated lucanohydrolase inding roteins (GBP) 1 and 2, that bind GLU I and CHN I both in vitro and when expressed in yeast cells. Sense as well as antisense transformants of tobacco carrying the GBP1 gene elaborated graft-transmissible acropetally moving signals that induced the downward curling of young leaves. This phenotype was associated with reduced starch, sucrose, and fructose accumulation; the formation of necrotic lesions; and, the induction of markers for the hypersensitive response. GBP1/2 are members of a conserved lant-specific yrin- repeat (PANK) family that includes proteins implicated in carbohydrate allocation, reactive oxygen metabolism, hypersensitive cell death, rapid elicitor responses, virus pathogenesis, and auxin signaling. The similarity in phenotype of PANK transformants and transformants altered in carbohydrate metabolism leads us to propose that PANK family members are multifunctional proteins involved in linking plant defense responses and carbohydrate metabolism.