Changes in pathogenesis-related proteins in pepper plants with regard to biological control of phytophthora blight with <Emphasis Type="Italic">Paenibacillus illinoisensis</Emphasis>

To evaluate the biocontrol effectiveness of chitinase-producing bacterium, Paenibacillus illinoisensis strain KJA-424 against pathogenic strain of Phytophthora capsici in pepper plants, growth response and kinetics of pathogen related (PR) proteins were estimated after inoculation with P. capsici (P), and with a combination of P. capsici and strain KJA-424 cell culture (P+A). Fresh weight and chlorophyll content in shoots at P+A-treated plants significantly increased by 23.4 and 34.2%, respectively after 7days of inoculation, compared to P-treated plants. Root mortality in P+A-treated plants was significantly reduced compared to P-treated plants. Seven days after inoculation, the activities of -1,3-glucanase, cellulase and chitinase in P-treated roots had decreased by 54.8, 36.5 and 52.8%, respectively, compared to P+A-treated roots, while those in P-treated leaves increased by 22.8, 36.3 and 23.8%, respectively, compared to those in P+A-treated leaves. The activities of -1,3-glucanase, cellulase and chitinase in roots are negatively correlated with root mortality. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, reduction of PR proteins in roots, and activates of PR proteins in leaves infected by P. capsici.     

Inoculation of Paenibacillus illinoisensis alleviates root mortality, activates of lignification-related enzymes, and induction of the isozymes in pepper plants infected by Phytophthora capsici

To investigate the variations of the enzymes responsible for lignification, after inoculation with Phytophthora capsici and/or Paenibacillus illinoisensis KJA-424, in relation to biocontrol of Phytophthora blight in pepper, roots of two-month-old plants were inoculated with P. capsici inoculation (P), and co-inoculation of P. capsici and P. illinoisensis cell cultures (P + A). Root mortality of pepper plants induced by inoculation of P. capsici was completely recovered by co-inoculation with antagonistic KJA-424. At day 7, peroxidase (POD) activity increased by 36.7% in P-treated roots but by 7.1% only in P + A-treated, compared with control. Polyphenol oxidase (PPO) activity increased for 3 days and then drastically decreased in P-treated roots but maintained a constant level in control and P + A-treated. At day 7, PPO activity in P-treated leaves decreased but recovered to the level of control in the P + A-treated. Three major POD isozymes (45, 53, and 114 kDa) were shown in P-treated roots, while two major (53 and 114 kDa) in control and P + A-treated, suggesting that the 45 kDa of POD was actively induced in P-treated roots but not induced in P + A-treated roots. A PPO isozyme of 80 kDa was induced in P-treated roots but not induced by co-treated with KJA-424. In leaves, the POD isozyme of 45 kDa appears to be systemically induced in P-treated only. The PPO isozyme of 80 kDa in leaves was not induced by pathogen challenge but recovered by co-inoculated with P. illinoisensis. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, activates of lignification-related enzymes and induction of the isozymes in pepper plants infected by P. capsici.     

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.     

Dynamics of Belonolaimus longicaudatus Parasitism on a Susceptible St. Augustinegrass Host

St. Augustinegrass (Stenotaphrum secundatum) cv FX-313 was used as a model laboratory host for monitoring population growth of the sting nematode, Belonolaimus longicaudatus, and for quantifying the effects of sting nematode parasitism on host performance in two samples of autoclaved native Margate fine sand with contrasting amounts of organic matter (OM = 7.9% and 3.8%). Following inoculation with 50 Belonolaimus longicaudatus per pot, nematodes peaked at a mean of 2,139 nematodes per pot 84 days after inoculation, remained stable through 168 days at 2,064 nematodes per pot, and declined at 210 days. The relative numbers of juveniles and adults demonstrated senescence after 84 days. Root dry weight of nematode-inoculated plants increased briefly to an apparent equilibrium 84 days after inoculation, whereas root weights of uninoculated controls continued to increase, exceeding those of inoculated plants from 84 to 210 days (P < 0.01). At 210 days, uninoculated plants had 227% the root dry weight of inoculated plants. Transpiration of FX-313 was reduced by nematodes (P < 0.0001) at 84 and 126 days after inoculation; reduction was first observed at 42 days and last observed 168 days after inoculation (P < 0.05). OM content affected all plant performance variables at multiple dates, and generally there were no inoculation x OM content interactions. OM content had no effect on nematode numbers per pot, although there was a slight (P < 0.05) increase in the number of nematodes per gram root dry weight in the low-OM soil compared with the high-OM soil.     

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.     

In vitro antagonism of cotton seedlings fungi and characterization of chitinase isozyme activities in Trichoderma harzianum

The antagonistic fungus Trichoderma harzianum is widely recognized as a potential biocontrol agent against several soil-borne plant pathogens. T. harzinum is rich source of chitinoltic enzymes. In vitro screening of 5 isolates of T. harzinum, one isolate of Chaetomium globosum and one isolate of Conetherium mentance, revealed that all of them had reduced growth area of Macrophomina phaseolina, Fusarium solaniand Rhizoctonia solani on PDA medium, significantly. The inhibition percentage ranged from 77.9 % to 55.9% for M. phaseolina and 59.2% to 40.4% for R. solani by T. harzinum and C. mentance, respectively. Inhibition for F. solani ranged from 76.5% to 55.7% by T. harzinum and C. globosum, respectively. Isozyme gel electrophoresis was used to assess chitinase activity secreted by selected isolates of T. harzinum under different pH degrees and temperatures. Obtained results indicated that activity of chitinase isozyme produced at 30 °C was higher than 15–20 °C for all tested isolates and activity of chitinase produced by isolates No. 4 and 5 of T. harzinum at pH (7–7.5) was higher than at pH 6, respectively.     

Suppression of Meloidogyne chitwoodi with Sudangrass Cultivars as Green Manure

Meloidogyne chitwoodi race 1 reproduced on Piper sudangrass (Sorghum bicolor (L.) Moench), 332 (sudangrass hybrid), and P855F and P877F (sorghum-sudangrass hybrids), but failed to reproduce efficiently on Trudan 8, Trudex 9 (sudangrass hybrids), and Sordan 79, SS-222, and Bravo II (sorghum-sudangrass hybrids). Meloidogyne chitwoodi race 2 behaved similarly and reproduced more efficiently on Piper, P855F, and P877F than on Trudan 8, Trudex 9, or Sordan 79. The mean reproductive factor for M. chitwoodi races on the poorer hosts ranged from <0.1 to 0.9 under greenhouse and field conditions. Meloidogyne hapla failed to reproduce on any of the cultivars tested. In the laboratory, leaves of each cultivar chopped and incorporated as green manure reduced the M. chitwoodi population in infested soil more than unamended or wheat green manure treatments. Trudan 8, although limited to the zone of incorporation, protected this zone from colonization of upward migrating second stage juveniles (J2) for up to 6 weeks. Leaves of Trudan 8 but not roots were effective against M. chitwoodi, and J2 appeared to be more sensitive than egg masses. Trudan 8 and Sordan 79 as green manure reduced M. chitwoodi in bucket microplots under field conditions.     

Effect of chitinase antisense RNA expression on disease susceptibility of Arabidopsis plants

Chitinases accumulate in higher plants upon pathogen attack are capable of hydrolyzing chitin-containing fungal cell walls and are thus implicated as part of the plant defense response to fungal pathogens. To evaluate the relative role of the predominate chitinase (class I, basic enzyme) of Arabidopsis thaliana in disease resistance, transgenic Arabidopsis plants were generated that expressed antisense RNA to the class I chitinase. Young plants or young leaves of some plants expressing antisense RNA had <10% of the chitinase levels of control plants. In the oldest leaves of these antisense plants, chitinase levels rose to 37–90% of the chitinase levels relative to vector control plants, most likely because of accumulation and storage of the enzyme in vacuoles. The rate of infection by the fungal pathogen Botrytis cinerea was measured in detached leaves containing 7–15% of the chitinase levels of control plants prior to inoculation. Antisense RNA was not effective in suppressing induced chitinase expression upon infection as chitinase levels increased in antisense leaves to 47% of levels in control leaves within 24 hours after inoculation. Leaves from antisense plants became diseased at a slightly faster rate than leaves from control plants, but differences were not significant due to high variability. Although the tendency to increased susceptibility in antisense plants suggests that chitinases may slow the growth of invading fungal pathogens, the overall contribution of chitinase to the inducible defense reponses in Arabidopsis remains unclear.     

Biological seed priming mitigates the effects of water stress in sunflower seedlings

The sunflower (Helianthus annuus L. cv. PAC 36) seedlings were inoculated with plant growth promoting rhizobacteria (PGPR), viz. Azotobacter chroococcum (A+), Bacillus polymyxa (B+), separately and in combination of the two (AB+). Relative water content and seedling growth were maximum in AB+ seedlings under control. Water stress significantly decreased the RWC, growth and dry mass of non-inoculated seedlings. However, inoculated seedlings maintained higher growth even under water stress. Pigments and protein contents decreased under water stress, but higher amount of the same was observed in stressed AB+ seedlings. Enhanced activity of nitrate reductase was recorded in AB+ seedlings with maximum in control. Water stress significantly decreased the nitrate reductase activity. A significant increase in the activity of superoxide dismutase (SOD) in leaves was recorded under water stress except in B+ with maximum increase in non-inoculated seedlings. Catalase (CAT) activity decreased in stressed non-inoculated seedlings while increased in the leaves of A+ and AB+ seedlings. Almost similar trends were recorded for both leaves and cotyledons. PGPR improved the water status in stressed seedlings and thereby physiological and biochemical parameters and thus ameliorated the severe effects of water stress.     

Dynamics of Concomitant Populations of Meloidogyne incognita and Criconemella xenoplax on Peach

The interaction between Meloidogyne incognita and Criconemella xenoplax on nematode reproduction and growth of Lovell peach was studied in field microlots and the greenhouse. Meloidogyne incognita suppressed reproduction of C. xenoplax in both field and greenhouse experiments. Tree growth, as measured by trunk diameter, was reduced (P ≤ 0.05) in the presence of M. incognita as compared with C. xenoplax of the uninoculated control trees 26 months following inoculation. A similar response regarding dry root weight was also detected in greenhouse-grown seedlings after 5 months. The presence of C. xenoplax did not affect Lovell tree growth. A synergistic effect causing a reduction (P ≤ 0.05) in tree growth was recorded 26 and 38 months following inoculation. The presence of M. incognita increased levels of malonyl-1-aminocyclopropane-1-carboxylic acid content in leaves of trees grown in field microplots 19 months after inoculaoon. Meloidogyne incognita appears to be a more dominant parasite than C. xenoplax on Lovell peach.     

Cucumis sativus L-type lectin receptor kinase (CsLecRK) gene family response to Phytophthora melonis, Phytophthora capsici and water immersion in disease resistant and susceptible cucumber cultivars

L-type lectin receptor kinase (LecRK) proteins are an important family involved in diverse biological processes such as pollen development, senescence, wounding, salinity and especially in innate immunity in model plants such as Arabidopsis and tobacco. Till date, LecRK proteins or genes of cucumber have not been reported. In this study, a total of 25 LecRK genes were identified in the cucumber genome, unequally distributed across its seven chromosomes. According to similarity comparison of their encoded proteins, the Cucumis sativus LecRK (CsLecRK) genes were classified into six major clades (from Clade I to CladeVI). Expression of CsLecRK genes were tested using QRT-PCR method and the results showed that 25 CsLecRK genes exhibited different responses to abiotic (water immersion) and biotic (Phytophthora melonis and Phytophthora capsici inoculation) stresses, as well as that between disease resistant cultivar (JSH) and disease susceptible cultivar (B80). Among the 25 CsLecRK genes, we found CsLecRK6.1 was especially induced by P. melonis and P. capsici in JSH plants. All these results suggested that CsLecRK genes may play important roles in biotic and abiotic stresses.     

Induction of Hydrolytic Enzymes in Brassica campestris in Response to Pathovars of Xanthomonas campestris

Inoculation of mature leaves of turnip (Brassica campestris) with the incompatible Xanthomonas campestris pv vitians resulted in the induction of β-1,3-glucanase and chitinase/lysozyme (CHL) activity. No increase in the basal activity of β-1,3-glucanase was observed after inoculation of leaves with heat- or rifampicin-killed X. c. vitians, Escherichia coli, or sterile water. Inoculation with the compatible X. campestris pv campestris resulted in a slower induction of glucanase than that seen with X. c. vitians. In contrast, all bacteria caused an induction of CHL activity. One major β-1,3-glucanase (molecular mass 36.5 kilodaltons, isoelectric point [pl] ~8.5) was purified from both inoculated and untreated leaves by ion-exchange chromatography. The enzyme degraded laminarin by an endo-glycolytic mechanism. Two major CHL isozymes (CHL 1 and CHL 2, molecular mass 30 kilodaltons and pl 9.4 and 10.2, respectively) were purified from X. c. vitians inoculated leaves by affinity chromatography on a chitin column followed by ion-exchange chromatography. Both enzymes degraded chitin by an endo-glycolytic mechanism although the ratio of lysozyme to chitinase specific activities for CHL 1 and CHL2 were different. The induction of CHL 1 was associated with the hypersensitive reaction caused by X. c. vitians whereas all other treatments induced largely CHL 2.     

Combined inoculation with Glomus intraradices and Rhizobium tropici CIAT899 increases phosphorus use efficiency for symbiotic nitrogen fixation in common bean (Phaseolus vulgaris L.)

This study compared the response of common bean (Phaseolus vulgaris L.) to arbuscular mycorrhizal fungi (AMF) and rhizobia strain inoculation. Two common bean genotypes i.e. CocoT and Flamingo varying in their effectiveness for nitrogen fixation were inoculated with Glomus intraradices and Rhizobium tropici CIAT899, and grown for 50 days in soil–sand substrate in glasshouse conditions. Inoculation of common bean plants with the AM fungi resulted in a significant increase in nodulation compared to plants without inoculation. The combined inoculation of AM fungi and rhizobia significantly increased various plant growth parameters compared to simple inoculated plants. In addition, the combined inoculation of AM fungi and rhizobia resulted in significantly higher nitrogen and phosphorus accumulation in the shoots of common bean plants and improved phosphorus use efficiency compared with their controls, which were not dually inoculated. It is concluded that inoculation with rhizobia and arbuscular mycorrhizal fungi could improve the efficiency in phosphorus use for symbiotic nitrogen fixation especially under phosphorus deficiency.     

A stress responsive alternative splicing mechanism in Citrus clementina leaves

Chitinases are often considered pathogenesis-related proteins since their activity can be induced by viral infections, fungal and bacterial cell wall components, and also by more general sources of stress such as wounding, salicylic acid, ethylene, auxins and cytokinins. In the present study, comparative proteomic analysis showed the defense-related acidic chitinase II to be specifically induced in Citrus clementina leaves infested by the two-spotted spider mite Tetranychus urticae or treated with MeJA. In parallel, changes in the mRNA profiles of two partially homologous chitinase forms were shown by RT-PCR. In particular, the appearance of an additional cDNA chitinase fragment in T. urticae-infested and MeJA-treated leaves was observed. This finding may indicate a specific regulatory mechanism of chitinase expression. We report evidence for alternative splicing in T. urticae-infested C. clementina, where a premature stop codon after the first 135 amino acids was introduced. We observed inducible chitinase activity after MeJA treatment, indicative of a rapid plant response to infestation. This work provides the first evidence of chitinase alternative splicing in C. clementina. In addition, the presence of the dual-band pattern for chitinase cDNA by RT-PCR may represent a suitable predictive marker for early diagnosis of plant biotic stress.     

Nodulation of Soybeans as Affected by Half-root Infection with Heterodera glycines

A split-root technique was applied to soybean, Glycine max (L.) Merr. cv. Lee 68, to characterize the nature of the nodulation suppression by race 1 of the soybean cyst nematode (SCN), Heterodera glycines. Root-halves of each split-root plant were inoculated with Rhizobium japonicum, and one root-half only was inoculated with various numbers of SCN eggs. Nodulation (indicated by nodule number, nodule weights, and ratio of nodule weight to root weight) and nitrogen-fixing capacity (indicated by rate of acetylene reduction) were systemically and variously suppressed on both root-halves of the split-root plant 5 weeks after half-root inoculation with 12,500 SCN eggs. Inoculation with 500 eggs caused this suppression only on the SCN-infected (+NE) root-half; nodulation on the companion uninfected (-NE) root-half was stimulated slightly. The +NE root-halves inoculated with 5,000 eggs were excised at 2-week intervals; nodulation on the remaining -NE root-halves was not different from that of the noninoculated control when measured 6 weeks after the SCN inoculation. Thus, the systemic suppression of nodulation was reversible upon the removal of the SCN. Similarly, application of various levels of KNO₃ to the -NE root-halves of the split-root plant did not alleviate the suppressed nodulation on the companion +NE root-halves, even though plant growth was much improved at certain levels of nitrogen (125 μg N/g soil). This indicated that the localized suppression of nodulation by SCN was caused by factors in addition to poor plant growth.     

Effect of nitrogen supply and Azospirillum brasilense Sp-248 on the response of wheat to seawater irrigation

Response of wheat to Azospirillum brasilense Sp-248 inoculation with different N-fertilizer levels using seawater irrigation was investigated. All inoculated treatments increased plant height, shoot and root dry weight, and tiller number in compared with uninoculated treatments. Yield parameters measured were also increased due to the inoculation. In terms of the effect of saline irrigation, there were no significant differences in growth and yield parameters in plants treated with tap water and others irrigated with 8.0% seawater concentration. This would indicate a relatively high tolerance of A. brasilense to saline irrigation and its ability to reduce the deleterious effects of saline on growth by increasing the plant’s adaptation. However, increasing the seawater concentration in the irrigation water to 16.0% significantly decreased all tested parameters. Inoculation treatments generally increased NPKCa contents and decreased sodium ratio of the grains in compared with the uninoculated treatments. Overall results clearly revealed that the Azospirillum inoculation saved about 20 units of N-fertilizer and that saving was made economically feasible by decreasing the chemical fertilizers needed, improving the nitrogen content and counteracting the effects of salinity.     

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.     

Interaction of Vesicular-arbuscular Mycorrhizal Fungi and Phosphorus with Meloidogyne incognita on Tomato

The influence of two vesicular-arbuscular mycorrhizal fungi and phosphorus (P) nutrition on penetration, development, and reproduction by Meloidogyne incognita on Walter tomato was studied in the greenhouse. Inoculation with either Gigaspora margarita or Glomus mosseae 2 wk prior to nematode inoculation did not alter infection by M. incognita compared with nonmycorrhizal plants, regardless of soil P level (either 3 μg [low P] or 30 μg [high P] available P/g soil). At a given soil P level, nematode penetration and reproduction did not differ in mycorrhizal and nonmycorrhizal plants. However, plants grown in high P soil had greater root weights, increased nematode penetration and egg production per plant, and decreased colonization by mycorrhizal fungi, compared with plants grown in low P soil. The number of eggs per female nematode on mycorrhizal and nonmycorrhizal plants was not influenced by P treatment. Tomato plants with split root systems grown in double-compartment containers which had either low P soil in both sides or high P in one side and low P in the other, were inoculated at transplanting with G. margarita and 2 wk later one-half of the split root system of each plant was inoculated with M. incognita larvae. Although the mycoorhizal fungus increased the inorganic P content of the root to a level comparable to that in plants grown in high P soil, nematode penetration and reproduction were not altered. In a third series of experiments, the rate of nematode development was not influenced by either the presence of G. margarita or high soil P, compared with control plants grown in low P soil. These data indicate that supplemental P (30 μ/g soil) alters root-knot nematode infection of tomato more than G. mosseae and G. margarita.