Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics

Molecular changes elicited by plants in response to fungal attack and how this affects plant–pathogen interaction, including susceptibility or resistance, remain elusive. We studied the dynamics in root metabolism during compatible and incompatible interactions between chickpea and Fusarium oxysporum f. sp. ciceri (Foc), using quantitative label‐free proteomics and NMR‐based metabolomics. Results demonstrated differential expression of proteins and metabolites upon Foc inoculations in the resistant plants compared with the susceptible ones. Additionally, expression analysis of candidate genes supported the proteomic and metabolic variations in the chickpea roots upon Foc inoculation. In particular, we found that the resistant plants revealed significant increase in the carbon and nitrogen metabolism; generation of reactive oxygen species (ROS), lignification and phytoalexins. The levels of some of the pathogenesis‐related proteins were significantly higher upon Foc inoculation in the resistant plant. Interestingly, results also exhibited the crucial role of altered Yang cycle, which contributed in different methylation reactions and unfolded protein response in the chickpea roots against Foc. Overall, the observed modulations in the metabolic flux as outcome of several orchestrated molecular events are determinant of plant's role in chickpea–Foc interactions.     

Induction of defense-related proteins in tomato roots treated with Pseudomonas fluorescens Pf1 and Fusarium oxysporum f. sp. lycopersici

Pseudomonas fluorescens isolate Pf1 was found to protect tomato plants from wilt disease caused by Fusarium oxysporum f. sp. lycopersici. Induction of defense proteins and chemicals by P. fluorescens isolate Pf1 against challenge inoculation with F. oxysporum f. sp. lycopersici in tomato was studied. Phenolics were found to accumulate in bacterized tomato root tissues challenged with F. oxysporum f. sp. lycopersici at one day after pathogen challenge. The accumulation of phenolics reached maximum at the 5^th day after pathogen challenge. In pathogen-inoculated plants, the accumulation started at the 2^nd day and drastically decreased 4 days after the pathogen inoculation. Activities of phenylalanine ammonia-lyase (PAL), peroxidase (PO) and polyphenol oxidase (PPO) increased in bacterized tomato root tissues challenged with the pathogen at one day after pathogen challenge and activities of PAL and PO reached maximum at the 4^th day while activity of PPO reached maximum at the 5^th day after challenge inoculation. Isoform analysis revealed that a unique PPO1 isoform was induced and PO1 and PPO2 isoforms were expressed at higher levels in bacterized tomato root tissues challenge inoculated with the pathogen. Similarly, -1,3 glucanase, chitinase and thaumatin-like proteins (TLP) were induced to accumulate at higher levels at 3-5 days of challenge inoculation in bacterized plants. Western blot analysis showed that chitinase isoform Chi2 with a molecular weight of 46 kDa was newly induced due to P. fluorescens isolate Pf1 treatment challenged with the pathogen. TLP isoform with molecular weight of 33 kDa was induced not only in P. fluorescens isolate Pf1-treated root tissues challenged with the pathogen but also in roots treated with P. fluorescens isolate Pf1 alone and roots inoculated with the pathogen. These results suggest that induction of defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restriction of invasion of F. oxysporum f. sp. lycopersici in tomato roots.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

Purification,characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one -1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The -1,3-glucanase (M_r = 36 kDa) and one of the chitinases (M_r = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (M_r = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic -1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibitied lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of -1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable. Finally, resitant (ILC 3279) and susceptible (ILC 1929) cultivars of chickpea showed no appreciable differences with regard to the time and amount of hydrolase accumulation after inoculation with spores of A. rabiei.Abbreviations AC acidic chitinase - BC basic chitinase - BG = basic -1,3-glucanase - CM-Chitin-RBV carboxymethylated-chitin-remazol brilliant violet - 2,4-D 2,4-dichlorophenoxyacetic acid - ILC international legume chickpea - M_r relative molecular mass - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis We thank the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financial support and ICARDA, Aleppo, Syria, for the provision of seed material. We also thank Dr. B. Fritig (Institut de Biologie Moléculaire des Plantes, CNRS, Straßbourg, France) and Dr. F. Meins, Jr. (Friedrich-Miescher-Institut, Basel, Switzerland) for their kind gifts of antibodies.     

Identification of defence proteins from the seed exudates of Cicer arietinum L. and its effect on the growth of Fusarium oxysporum f.sp. ciceri

Germinating seeds tend to release a variety of proteins into their surrounding surfaces; some of which have an inhibitory action against plant pathogens. The aim of this study was to investigate and identify defence proteins present in the exudates from water-imbibed and chitosan-imbibed (0.1% w/v) seeds of chickpea (Cicer arietinum L). Chickpea seeds imbibed in chitosan released a higher amount of proteins in the exudate when compared to the seeds imbibed in water. The obtained exudates were analysed in regard to specific protein activities by enzymatic assays and SDS-PAGE analysis. Results showed that the exude obtained from chickpea seeds imbibed in chitosan solution exhibited a new isoform of chitinase, chitosanase and protease inhibitors. These exudates also have an “in vitro” inhibitory effect on the growth of the fungus, Fusarium oxysporum f.sp. ciceri. Our results suggest that seed exudates protect seeds during their germination from soil pathogens.     

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.     

Potential of microsatellites to distinguish four races of Fusarium oxysporum f. sp. ciceri prevalent in India

Fusarium oxysporum f. sp. ciceri, the causal agent of chickpea wilt, is an important fungal pathogen in India. Thirteen oligonucleotide probes complementary to microsatellite loci, in combination with 11 restriction enzymes, were used to assess the potential of such markers to study genetic variability in four Indian races of the pathogen. Hybridisation patterns, which were dependent upon both the restriction enzyme and oligonucleotide probe used, revealed the presence of different repeat motifs in the F. oxysporum f. sp. ciceri genome. Among the restriction enzymes used, hexa-cutting enzymes were more informative than tetra- and penta-cutting enzymes, whereas tetranucleotide and trinucleotide repeats yielded better hybridisation patterns than dinucleotide repeats. Dependent upon the levels of polymorphism detected, we have identified (AGT)₅, (ATC)₅ and (GATA)₄ as the best fingerprinting probes for the F. oxysporum f. sp. ciceri races. The distribution of microsatellite repeats in the genome revealed races 1 and 4 to be closely related at a similarity index value of 76.6%, as compared to race 2 at a similarity value of 67.3%; race 3 was very distinct at a similarity value of 26.7%. Our study demonstrates the potential of oligonucleotide probes for fingerprinting and studying variability in the F. oxysporum f. sp. ciceri races and represents a step towards the identification of potential race diagnostic markers. Received: 12 March 2000 / Accepted: 14 April 2000     

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.     

Interaction of Fusarium oxysporum f. sp. ciceri and Meloidogyne javanica on Cicer arietinum

Interaction of Meloidogyne javanica and Fusarium oxysporum f. sp. ciceri was studied on Fusarium wilt-susceptible (JG 62 and K 850) and resistant (JG 74 and Avrodhi) chickpea cultivars. In greenhouse experiments, inoculation of M. javanica juveniles prior to F. oxysporum f. sp. ciceri caused greater wilt incidence in susceptible cultivars and induced vascular discoloration in roots of resistant cultivars. Nematode reproduction was greatest (P = 0.05) at 25 °C. Number of galls and percentage of root area galled increased when the temperature was increased from 15 °C to 25 °C. Wilt incidence was greater at 20 °C than at 25 °C. Chlorosis of leaves and vascular discoloration of plants did not occur at 15 °C. The nematode enhanced the wilt incidence in wilt-susceptible cultivars only at 25 °C. Interaction between the two pathogens on shoot and root weights was significant only at 20 °C, and F. o. ciceri suppressed the nematode density at this temperature. Wilt incidence was greater in clayey (48% clay) than in loamy sand (85% sand) soils. The nematode caused greater plant damage on loamy sand than on clayey soil. Fusarium wilt resistance in Avrodhi and JG 74 was stable in the presence of M. javanica across temperatures and soil types.     

The Effect of Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense on Induction of Defense Enzymes and Phenolics in Banana

The effect of Pseudomonas fluorescens treatment and Fusarium oxysporum f. sp. cubense inoculation on induction of phenylalanine ammonia-lyase (PAL), peroxidase (POX), chitinase, -1,3-glucanase and accumulation of phenolics in banana (Musa sp.) was studied. When banana roots were treated with P. fluorescens strain Pf10, a two-fold increase in phenolic content in leaf tissues was recorded 3 – 6 d after treatment. Challenge inoculation with F. oxysporum, the wilt pathogen, steeply increased the phenolic content in P. fluorescens-treated banana plants. Significant increase in POX activity was detected 6 – 9 d after P. fluorescens treatment. PAL, chitinase and -1,3-glucanase activities increased significantly from 3 d after P. fluorescens treatment and reached the maximum 6 d after treatment. Challenge inoculation with F. oxysporum further increased the enzyme activities. These results suggest that the enhanced activities of defense enzymes and elevated content of phenolics may contribute to bioprotection of banana plants against F. oxysporum.     

Evaluation of Streptomyces spp. and Bacillus spp. for biocontrol of Fusarium wilt in chickpea (Cicer arietinum L.)

A study was carried out to test direct and indirect antagonistic effect against Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (FOC), and plant growth-promoting (PGP) traits of bacteria isolated from rhizosphere soils of chickpea (Cicer arietinum L.). A total of 40 bacterial isolates were tested for their antagonistic activity against FOC and of which 10 were found to have strong antagonistic potential. These were found to be Streptomyces spp. (five isolates) and Bacillus spp. (five isolates) in the morphological and biochemical characterisation and 16S rDNA analysis. Under both greenhouse and wilt sick field conditions, the selected Streptomyces and Bacillus isolates reduced disease incidence and delayed expression of symptoms of disease, over the non-inoculated control. The PGP ability of the isolates such as nodule number, nodule weight, shoot weight, root weight, grain yield and stover yield were also demonstrated under greenhouse and field conditions over the non-inoculated control. Among the ten isolates, Streptomyces sp. AC-19 and Bacillus sp. BS-20 were found to have more potential for biocontrol of FOC and PGP in chickpea. This investigation indicates that the selected Streptomyces and Bacillus isolates have the potential to control Fusarium wilt disease and to promote plant growth in chickpea.     

Exploring Crop–Microbiome Interactions Towards Improving Symbiotic Performance of Chickpea (Cicer arietinum) Cultivars Using Cyanobacterial Inoculants

The significance of integrated nutrient management practices is well established in improving the productivity of chickpea (Cicer arietinum); however, the effects of the inoculation of cyanobacterial inoculants on nodule metabolism, microbiome and associated genes are less explored. In the present investigation, cyanobacterium Anabaena laxa (A. laxa) and biofilm developed using Anabaena torulosa, with Mesorhizobium ciceri as a partner (An-M. ciceri), were evaluated along with Mesorhizobium ciceri (M. ciceri) alone, in three chickpea cultivars. Microbial inoculation led to 40–70% enhancement in nitrogen fixation, leghaemoglobin and ureide content, and two- to threefold increment in nitrate reductase and phosphoenolpyruvate carboxylase activity of the nodules. An enhancement of 30–50% in the soil available macro- and micronutrients and plant growth attributes was also observed. A significant correlation between the soil microbiological and plant parameters was recorded, particularly in relation to the nitrogen dynamics. Increases in the leghaemoglobin content in nodules due to An-M. ciceri, A. laxa and M. ciceri ranged from 18 to 40%, particularly in chickpea cv. BG372 in which 60–80% enhancement was recorded. Whereas the nifH gene copies in the nodule tissues ranged from 5.00 × 10⁶ to 3.35 × 10⁷ g⁻¹, the application of A. laxa led to higher abundances of nifH gene copies in desi chickpea cv. BG372 and kabuli BG1053 cultivars. An-M. ciceri, followed closely by A. laxa, was the top-ranking treatment, and chickpea cv. BG372 was the best performing cultivar; An-M. ciceri—chickpea cv. BG372 proved to be the superior combination for higher plant growth and soil nutrient-related traits.

     

Biologic Control Ability of Plant Growth–Promoting Paenibacillus lentimorbus NRRL B-30488 Isolated from Milk

A plant growth–promoting Paenibacillus lentimorbus NRRL B-30488 (B-30488) was isolated from cows’ milk. Bacterial colonization and growth responses of different plant species after inoculation with B-30488 were evaluated in a controlled environment and in microplot assays. Survival and colonization of B-30488 in the phytosphere of plants and soil was monitored using a chromosomally located rifampicin-marked mutant B-30488 (B-30488R). The strain showed variable ability to invade plants. The interaction between B-30488R and Fusarium oxysporum f. sp. ciceri was studied by scanning electron microscopy. Chitinase and β-1,3-glucanase enzymes were produced when B-30488R was grown in the presence of colloidal chitin as sole carbon source. Deliberate dilution of B-30488R with field soil offers a reliable process for decreasing the cost of bacterial inoculants in developing countries. Seed treatment of chickpea demonstrated significantly (P = 0.05) greater seedling mortality in nonbacterized compared with bacterized seedlings. Bacterization significantly (P = 0.05) improved seed germination, plant height, number of pods/plant^–1, and seed dry weight.     

Saprophytic colonization ofFusarium oxysporum f. sp.ciceri in soil

Competitive saprophytic colonization (CSC) of soybean and chickpea stem pieces byFusarium oxysporum f.sp.ciceri increased with the increase in inoculum density in inoculum soil mixtures. The colonization was higher even at loeer concentration of inoculum. Progressive dilutions of autoclaved soils with unsterilized soil decreased the CSC. Lower temperatures favoured the colonization in both red sandy loam and black soils. Maximum colonization occurred at 40°C indicating an inverse relation between colonization and temperature.     

Host-specific Fusarium oxysporum Causes Wilt of Jojoba

Jojoba [Simmondsia chinensis (Link) Schneider] plantations in Israel originated from vegetative propagation, planted during 1991–92, have shown symptoms of wilting and subsequent death. Verticillium dahliae was only rarely isolated from these plants and artificial inoculation showed only mild disease symptoms. Fusarium oxysporum caused severe chlorosis, desiccation, defoliation and wilt in leaves of jojoba plants, resulting in plant death. Recovery of the fungus from artificially inoculated stem cuttings and seedlings showed for the first time that F. oxysporum was the primary pathogen. Inoculated cuttings exhibited wilt within 3 weeks, while in seedlings wilt occurred 10–24 weeks after inoculation. Seedlings and cuttings of jojoba which were inoculated with other Fusarium isolates originating from different crops (F. oxysporum f. sp. vasinfectum from cotton, F. oxysporum f. sp. dianthi from carnation, F. oxysporum f. sp. lycopersici from tomato and F. oxysporum f. sp. basilicum from basil) did not develop symptoms. Moreover, cotton, tomato, melon and cucumber seedlings inoculated with several virulent F. oxysporum isolates from jojoba did not show any symptoms of wilt or defoliation. These results indicate a high degree of specificity of the Fusarium isolates from jojoba; therefore, it is suggested that this isolate be defined as F. oxysporum f. sp. simmondsia.     

Abscisic Acid–Responsive 18 (CaABR18) Protein from Chickpea Inhibits the Growth of the Wilt-Causing Fusarium oxysporum f. sp. ciceri Race1

Multigene family pathogenesis-related-10 (PR-10) proteins are indispensable for initiation of plant defense reactions upon pathogen attack. Here, we report the isolation and differential induction of Cicer arietinum L. ABR18 (CaABR18) gene in susceptible and resistant chickpea upon exposure to Fusarium oxysporum f. sp. ciceri Race1 (Foc1). Further, sequence analysis and structural studies confirmed that CaABR18 protein possesses conserved glycine-rich P-loop motif and Betv 1 domain, which are common to many PR-10 family proteins. CaABR18 gene was found to be expressed in all the developing organs, with higher abundance in the mature leaves. Foc1 inoculation resulted in higher expression of CaABR18 gene in the resistant chickpea compared with susceptible one. CaABR18 protein induction was also observed by salicylic acid (SA) or abscisic acid (ABA) treatment. Biochemical analysis was performed using in vitro purified histidine-tagged recombinant ABR18 protein. Purified recombinant protein exhibits in vitro RNase and DNase activities. Application of recombinant ABR18 protein increases PI/SYTOX green uptake and nuclear disintegration and suppresses the growth of Foc1 hyphae in vitro. Agrobacterium-mediated transient expression of ABR18-YFP triggers reactive oxygen species (ROS) formation and cell death in Nicotiana benthamiana leaves. The fusion protein is shown to be targeted to the host nucleus. Taken together, our results revealed that CaABR18 imparts Fusarium resistance in chickpea by RNA/DNA degradation within host cells leading to programmed cell death (PCD) and also shows antifungal activity through its proper internalization, increasing membrane permeability and nuclear disintegration of Foc1.

     

Differential effects of coinoculations with Pseudomonas jessenii PS06 (a phosphate-solubilizing bacterium) and Mesorhizobium ciceri C-2/2 strains on the growth and seed yield of chickpea under greenhouse and field conditions

In the course of a project carried out in two regions of Spain, Castilla y León and Andalucía, aiming to find useful biofertilizers for staple grain-legumes, an efficient rhizobia nodulating chickpea (termed as C-2/2) and a powerful in vitro phosphate-solubilizing bacterial strain (termed as PS06) were isolated. Analyses of their 16S rDNA sequence indicated that they belong to the bacterial species Mesorhizobium ciceri and Pseudomonas jessenii, respectively. Greenhouse and field experiments were carried out in order to test the effect of single and dual inoculations on chickpea (ecotype ILC-482) growth. Under greenhouse conditions, plants inoculated with Mesorhizobium ciceri C-2/2 alone had the highest shoot dry weight. The inoculation treatment with P. jessenii PS06 yielded a shoot dry weight 14% greater than the uninoculated control treatment, but it was not correlated with shoot P contents. However, the co-inoculation of C-2/2 with PS06 resulted in a decrease in shoot dry weight with respect to the inoculation with C-2/2 alone. Under field conditions, plants inoculated with M. ciceri C-2/2, in single or dual inoculation, produced higher nodule fresh weight, nodule number and shoot N content than the other treatments. Inoculation with P. jessenii PS06 had no significant effect on plant growth. However, the co-inoculation treatment ranked the highest in seed yield (52% greater than the uninoculated control treatment) and nodule fresh weight. These data suggest that P. jessenii PS06 can act synergistically with M. ciceri C-2/2 in promoting chickpea growth. The contrasting results obtained between greenhouse and field experiments are discussed.     

Potential role of rhizobia to enhance chickpea-growth and yield in low fertility-soils of Tunisia

Plant growth-promoting rhizobacteria are bacteria that improve plant growth and reduce plant pathogen damages. In this study, 100 nodule bacteria were isolated from chickpea, screened for their plant growth-promoting (PGP) traits and then characterised by PCR-RFLP of 16 S rDNA. Results showed that most of the slow-growing isolates fixed nitrogen but those exhibiting fast-growth did not. Fourteen isolates solubilized inorganic phosphorus, 16 strains produced siderophores, and 17 strains produced indole acetic acid. Co-culture experiments identified three strains having an inhibitory effect against Fusarium oxysporum, the primary pathogenic fungus for chickpea in Tunisia. Rhizobia with PGP traits were assigned to Mesorhizobium ciceri, Mesorhizobium mediterraneum, Sinorhizobium meliloti and Agrobacterium tumefaciens. We noted that PGP activities were differentially distributed between M. ciceri and M. mediterraneum. The region of Mateur in northern Tunisia, with clay–silty soil, was the origin of 53% of PGP isolates. Interestingly, we found that S. meliloti and A. tumefaciens strains did not behave as parasitic nodule-bacteria but as PGP rhizobacteria useful for chickpea nutrition and health. In fact, S. meliloti strains could solubilize phosphorus, produce siderophore and auxin. The A. tumefaciens strains could perform the previous PGP traits and inhibit pathogen growth also. Finally, one candidate strain of M. ciceri (LL10)—selected for its highest symbiotic nitrogen fixation and phosphorus solubilization—was used for field experiment. The LL10 inoculation increased grain yield more than three-fold. These finding showed the potential role of rhizobia to be used as biofertilizers and biopesticides, representing low-cost and environment-friendly inputs for sustainable agriculture.

     

Chitinase isozymes induced by TYMV and <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> during compatible and incompatible interaction with <Emphasis Type="Italic">Brassica napus</Emphasis>

Accumulation of extracellular chitinases in Brassica napus plants infected with Turnip yellow mosaic virus (TYMV) and fungal pathogen Leptosphaeria maculans was studied in both compatible and incompatible interaction. Analysis of apoplast fluid by means of non-denaturing anodic and cathodic PAGE followed by in-gel detection of chitinase activity revealed a number of chitinase isozymes. TYMV induced 8 acidic and 4 basic isozymes in a systemic way. Except for one acidic and one basic isozyme, all other chitinases were also constitutively present in low amounts in mock inoculated control. In TYMV systemically infected plants, chitinases were detected in leaves expressing symptoms as well as in symptomless ones. Both virulent and avirulent L. maculans isolates induced production of chitinase isozymes in cotyledons in a time dependent manner. Some of them were present in plants constitutively and their content increased after inoculation. Three of five acidic and two of three basic isozymes responded to L. maculans infection. Chitinases started to accumulate before symptom appearance. First two acidic isozymes were detected 24 h after inoculation. The difference between compatible and incompatibe interaction reflected two basic isozymes.