M. E. Candela

Evaluation of induction of systemic resistance in pepper plants (Capsicum annuum) to Phytophthora capsici using Trichoderma harzianum and its relation with capsidiol accumulation.

The effect of pepper seed and root treatments with Trichoderma harzianum spores on necrosis caused in stems by Phytophthora capsici inoculation and on the course of capsidiol accumulation in the inoculated sites were studied. The results indicate that seed treatments significantly reduced stem necrosis, which fell by nearly a half compared with the values observed in plants grown from non-treated seeds. Necrosis was also reduced in plants whose roots were drenched with various doses of T. harzianum spores, although the extent of necrosis was not correlated with the dose used. Attempted isolation of P. capsici and T. harzianum from the zones immediately contiguous with the necrotic zones revealed the presence of the former but not of the latter, suggesting that there was no direct contact between them in the zones of isolation, which means that there was no competition for space. The percentage of P. capsici isolated 9 days after inoculation was greater in non-treated inoculated plants than in treated inoculated plants. These results suggest that T. harzianum, introduced into the subterranean part of the plant, induces a systemic defense response against P. capsici in the upper part of the plant. Analysis of capsidiol in the stems of treated inoculated plants by the end of the sixth day after inoculation, revealed that its concentration was more than seven-fold greater than in non-treated and inoculated plants, while after 9 days, the concentration of capsidiol decreased in the treated inoculated plants and increased in the non-treated inoculated plants. The high concentration of capsidiol detected in treated and inoculated stems after 6 days might be one of the contributing factors, but not necessarily the main factor, in delaying lesion development in the stems of pepper plants.

Effect of chitin on biological control activity of Bacillus spp. and Trichoderma harzianum against root rot disease in pepper (Capsicum annuum) plants

Two bacterial isolates and one strain of Trichoderma harzianum were tested alone and in combination with chitin for efficacy in control of root rot disease caused by Phytophthora capsici and Rhizoctonia solani in pepper plants under greenhouse conditions. These bacteria (Bacillus subtilis HS93 and B. licheniformis LS674) were isolated from repeatedly washed roots of pepper plants. In in vitro assays, HS93, LS674 and T. harzianum were antagonistic against P. capsici and R. solani and produced high levels of chitinase. Seed treatment and root drenching with bacterial suspensions of HS93 with 0.5% chitin was more effective against Phytophthora and Rhizoctonia root rot than addition of the organisms without chitin. LS674 and T. harzianum reduced Rhizoctonia but not Phytophthora root rot. In two greenhouse tests, seed treatment and root drenching with HS93 amended with chitin enhanced its biocontrol activity against P. capsici but not on R. solani. The effects of LS674 and T. harzianum against R. solani were significantly enhanced when they were used as suspensions with 0.5% chitin for root drenching, but this had no effect on P. capsici. In both greenhouse experiments, the use of 0.5% chitin alone for root drenching reduced Rhizoctonia root rot. Reduction of root rot disease was accompanied by increased yield. These results show that the antagonistic activity of HS93, LS674 and T. harzianum may be stimulated by chitin resulting in significant improvements in their effectiveness against pathogens.

Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells by Phytophthora capsici

Summary Cell suspension cultures of three varieties of Capsicum annuum L., each with a different degree of sensitivity to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate with a hypersensitive reaction. They showed the synthesis or accumulation of PR-proteins with peroxidase (EC 1.11.1.7) activity and the accumulation of lignin-like polymer (as measured by derivatization with thioglycolic acid). The cultivation medium was optimised for both plant and fungus growth in order to avoid any stress during their combination. The resistant pepper variety, Smith-5, showed a more intense response to the elicitor preparations than the sensitive varieties, Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate. After elicitation, the cell walls thickened through the accumulation of lignin, as can be observed by staining microscope preparations with methylene blue. Elicitation also reduced the level of total peroxidase activity in the susceptible varieties, while such activity increased in resistant varieties, and was accompanied by de novo expression of acidic peroxidase isoenzymes in the extracellular and cell wall fractions. Of note was the PR protein of pI 5.7 showing peroxidase activity, which was induced by both elicitor types in the elicited cell suspensions of the resistant variety alone, making it a marker of resistance. The increases in the activity of these peroxidases in the resistant variety are in concordance with the accumulation of lignin observed 24 h after inoculation by both elicitors from the fungus. The possible role of these isoenzymes in lignin biosynthesis, used to reinforce the cell walls against fungal penetration of the cells, is discussed. These results are in accordance with those previously observed in plant stem sections.

Selecting bacterial strains for use in the biocontrol of diseases caused by Phytophthora capsici and Alternaria alternata in sweet pepper plants

More than 500 isolates of bacteria were obtained from the aerial part and rhizosphere of sweet pepper (Capsicum annuum L.) plants harvested from different places in the Region of Murcia (Spain). The isolates were purified and assayed in vitro against Phytophthora capsici and Alternaria alternata. Sixty isolates (12 %) produced an inhibition zone against at least one of the pathogens, while ten had a strongly inhibitory effect on both pathogens assayed. Microscopic observation of interactions zone showed cell vacuolisation, hyphae lysis and spilling of cytoplasm content of the pathogens in the culture media. These ten isolates were then chosen for biocontrol of Phytophthora root rot and Alternaria leaf spots of pepper plants in vivo. Four of them denominated HS93, LS234, LS523 and LS674 reduced P. capsici root rot by 80, 51, 49 and 54 %, respectively, and A. alternata leaf spots by 54, 74, 62 and 53 %. HS93 belongs to the genus Bacillus and probably the species subtilis, while LS234, LS523 and LS674 belong to the genus Bacillus and probably the species licheniformis. Dry mass of plants treated with these bacteria was significantly higher than that of non-treated and inoculated plants.

Isoperoxidase Involvement in the Resistance of Capsicum annuum to Infection by Cucumber Mosaic Virus

Summary Cucumber mosaic virus (CMV) infection of Capsicum annuum leaves provokes a strong increase in soluble peroxidase activity. Isoelectrofocusing of the peroxidase activity in polyacrylamide gel patterns, after staining the soluble protein fractions from both healthy and infected leaves with benzidine/H 2 O 2 , revealed the presence of two acidic and one basic isoperoxidases, the increase in peroxidase activity being mainly due to the former. Staining of peroxidase activity in the gels with coniferyl alcohol/H 2 O 2 showed that acidic isoenzymes were more capable of catalyzing coniferyl alcohol oxidation than basic isoenzymes. Native polyacrylamide gel electrophoresis of the soluble fraction showed that the increase in peroxidase isoenzymes. CMV elicitation of the acidic isoperoxidase through its involvement in the lignification process might be considered as a part of the defense mechanism of Capsicum annuum against viral infection, lignin deposition on plasmodesmata could act as a physical barrier against the cell-to-cell advance of viral particles.

Pepper morphological traits related with resistance to Phytophthora capsici

Inheritance of 10 morphological and quantitative traits related to plant and fruit development and resistance to the pathogen Phytophthora capsici was studied in an intraspecific cross between a non-pungent, susceptible Capsicum annuum parent (cv. Americano) and a wild, pungent and resistant line (Serrano Criollo de Morelos-334). Data were obtained from the segregation of 166 F2 plants and 50 F3 plants in four years. Three of the traits analyzed (necrosis length, leaf width and leaf length) exhibited a transgressive segregation. A multiple linear regression analysis was applied in order to establish a relationship between necrosis length and some of the morphological traits measured such as length and width of leaf, length, diameter and mass of fruit, capsaicin content in fruits, and presence of hair on leaves and stems. The results identified a linear dependence between necrosis length (as an inverse measurement of resistance) and leaf width, fruit diameter and hair presence in the stem. Pungency was not related with resistance.

β-1,3-glucanase and chitinase as pathogenesis-related proteins in the defense reaction of twoCapsicum annuum cultivars infected with cucumber mosaic virus

Pathogenesis-related (PR) proteins from pepper (Capsicum annuum L.) cv. Americano (tolerant) and cv. Smith-5 (sensitive), both elicited by infection with cucumber mosaic virus (CMV), were assayed for chitinase and glucanase activities. Two basic PR-proteins, Mr 49.0 and 28.0 kD, were elicicited from the intracellular fraction (INTRA-F) of both cvs. by CMV infection, while four acidic Mr 15, 19, 36 and 40 kD and two basic Mr 21.2 and 24 kD PR-proteins were elicited from the intercellular fluid (IF) of cv. Americano leaves. Five acidic Mr 21.5, 23.2, 24.4, 25.2 and 36 kD and five basic Mr 23.3, 26, 28.8, 30 and 32.3 kD PR proteins were elicited from the IF of cv. Smith-5. Isoelectric focusing (IEF) of the IF and the INTRA-F proteins revealed the occurrence, in both pepper cultivars, of one acidic Mr 36 kD and one basic Mr 25 kD PR-protein with glucanase activity. After native-PAGE for acidic proteins, the acidic PR-protein of Rf 0.7 and Mr 36 kD present in the IF of both pepper cvs. showed glucanase activity. Native-PAGE for basic proteins of the INTRA-F showed the presence of one band (Rf 0.61, Mr 25 kD) common to both cvs. and two additional bands (Rf 0.49, Mr 26 kD and Rf 0.79, Mr 33 kD) in the cv. Americano with glucanase activity. The specificity shown by the basic PR-proteins suggests glucanase activity is involved in the mechanisms of resistance to CMV in the cv. Americano. There was no difference in chitinase isoform patterns between the two pepper cultivars analyzed. After IEF of the IF proteins, one acidic chitinase isoform was detected. Native-PAGE separation of the IF showed one band (Mr 30 kD) with chitinase activity. Chitinase activity was not detected in the INTRA-F of either cultivar.

Identification of different nutritional status groups in institutionalized elderly people by cluster analysis

OBJECTIVES To apply a cluster analysis to groups of individuals of similar characteristics in an attempt to identify undernutrition or the risk of undernutrition in this population. METHODS DESIGN A cross-sectional study. SETTING Seven public nursing homes in the province of Murcia, on the Mediterranean coast of Spain. PARTICIPANTS 205 subjects aged 65 and older (131 women and 74 men). MEASUREMENTS Dietary intake (energy and nutrients), anthropometric (body mass index, skinfold thickness, mid-arm muscle circumference, mid-arm muscle area, corrected arm muscle area, waist to hip ratio) and biochemical and haematological (serum albumin, transferrin, total cholesterol, total lymphocyte count). Variables were analyzed by cluster analysis. RESULTS The results of the cluster analysis, including intake, anthropometric and analytical data showed that, of the 205 elderly subjects, 66 (32.2%) were over - weight/obese, 72 (35.1%) had an adequate nutritional status and 67 (32.7%) were undernourished or at risk of undernutrition. The undernourished or at risk of undernutrition group showed the lowest values for dietary intake and the anthropometric and analytical parameters measured. CONCLUSIONS Our study shows that cluster analysis is a useful statistical method for assessing the nutritional status of institutionalized elderly populations. In contrast, use of the specific reference values frequently described in the literature might fail to detect real cases of undernourishment or those at risk of undernutrition.

Soluble pathogenesis-related proteins ofCapsicum annuum leaves induced by cucumber mosaic virus infection

Eight major pathogenesis related (PR) proteins were found in a soluble extract of cucumber mosaic virus (CMV) infectedCapsicum annuum leaves. None of them was present in the soluble extract of sham-inoculated controls. The proteins were detected by two-dimensional polyacrylamide gel electrophoresis using (2D-PAGE)PhastSystem, native-PAGE in the first dimension and SDS-PAGE in the second. Two major acidic PR-proteins were identified on the basis of their relative molecular mass (Mr), PR1 of 15.3 kD and PR2 of 29 kD. Six proteins were basic and were identified as PRla of 15.9 kD, PRlb of 15.0 kD, PRlc of 15.9 kD, PR2 of 27.0 kD, PR3 of 36.3 kD and PR4 of 48.8 kD.