Epigenetic responses to Phytophthora citrophthora gummosis in citrus.

Studies show that DNA methylation is associated with plant immunity but little is known as to how this epigenetic mechanism assists plants in adjusting their responses to biotic stress, especially when interacting with an hemibiotrophic pathogen such as citrus Phytophthora. The aim of the present study was to assess the effects of scion-rootstock interaction on plant resistance to P. citrophthora infection and DNA methylation patterns in 'Pera' sweet orange and 'Tahiti' acid lime grafted onto 'Rangpur' lime and 'Tropical' sunki rootstocks reinoculated with P. citrophthora. Results showed that reinoculated plants of the 'Pera' sweet orange/'Rangpur' lime and 'Tahiti' acid lime/'Tropical' sunki combinations with more and less sensitive varieties to Phytophthora, presented smaller stem lesions and increased frequency of full methylation and hemimethylation rates, compared to inoculated plants. In contrast, 'Tahiti' acid lime/'Rangpur' lime, two highly sensitive varieties, and 'Pera'/'Tropical' sunki, two much less sensitive varieties, showed high increases in the frequency of hemimethylation and non-methylation levels. Results suggest that in citrus, both the scion-rootstock interaction and DNA methylation affect the response to P. citrophthora infection. Reinoculated plants, depending on the combination, showed changes in intracellular hyphae growth through the formation of sets of fibers and crystal accumulation in the periderm, cortex, and phloem. In addition, starch grain concentration was higher in reinoculated plants in comparison to inoculated plants. These findings support the assumption that DNA methylation is a plant defense mechanism and therefore may be exploited to improve the response of plants to the gummosis of P. citrophthora in citrus.

Illumina-MiSeq analysis of fungi in acid lime roots reveals dominance of Fusarium and variation in fungal taxa.

A study was conducted to analyze fungal diversity in the roots of acid lime (Citrus aurantifolia) collected from Oman, a semi-arid country located in the South Eastern part of the Arabian Peninsula. MiSeq analysis showed the Ascomycota and Sordariomycetes were the most abundant phylum and class in acid lime roots, respectively. Glomeromycota, Basidiomycota and Microsporidia were the other fungal phyla, while Glomeromycetes and some other classes belonging to Ascomycota and Basidiomycota were detected at lower frequencies. The genus Fusarium was the most abundant in all samples, making up 46 to 95% of the total reads. Some fungal genera of Arbuscular mycorrhizae and nematophagous fungi were detected in some of the acid lime roots. Analysis of the level of fungal diversity showed that no significant differences exist among groups of root samples (from different locations) in their Chao richness and Shannon diversity levels (P < 0.05). Principle component analysis of fungal communities significantly separated samples according to their locations. This is the first study to evaluate fungal diversity in acid lime roots using high throughput sequencing analysis. The study reveals the presence of various fungal taxa in the roots, dominated by Fusarium species and including some mycorrhizae and nematophagous fungi.

Witch's Broom Disease of Lime (Candidatus Phytoplasma aurantifolia): Identifying High-Risk Areas by Climatic Mapping.

Biological invasions of vectorborne diseases can be devastating. Bioclimatic modeling provides an opportunity to assess and predict areas at risk from complex multitrophic interactions of pathogens, highlighting areas in need of increased monitoring effort. Here, we model the distribution of an economically critical vectorborne plant pathogen 'Candidatus Phytoplasma aurantifolia', the etiological agent of Witches' Broom Disease of Lime. This disease is a significant limiting factor on acid lime production (Citrus aurantifolia, Swingle) in the Middle East and threatens its production globally. We found that temperature, humidity, and the vector populations significantly determine disease distribution. Following this, we used bioclimatic modeling to predict potential novel sites of infections. The model outputs identified potential novel sites of infection in the citrus producing regions of Brazil and China. We also used our model to explore sites in Oman where the pathogen may not be infectious, and suggest nurseries be established there. Recent major turbulence in the citrus agricultural economy has highlighted the importance of this work and the need for appropriate and targeted monitoring programs to safeguard lime production.

Dynamics of Huanglongbing-associated Bacterium Candidatus Liberibacter asiaticus in Citrus aurantifolia Swingle (Mexican Lime).

BACKGROUND: The bacterial disease citrus huanglongbing (HLB), associated with "Candidatus Liberibacter asiaticus" (C.Las) has severely impacted the citrus industry, causing a significant reduction in production and fruit quality. In the present study, it was monitored the C.Las population dynamics in symptomatic, HLB-positive Mexican lime trees (Citrus aurantifolia Swingle) in a tropical, citrus-producing area of Mexico. The objective of this study was to identify the dynamics of the population of huanglongbing-associated bacterium Candidatus Liberibacter asiaticus and its insect vector in Citrus aurantifolia Swingle (Mexican lime). MATERIALS AND METHODS: Leaf samples were collected every 2 months over a period of 26 months for quantification of bacterial titers and young and mature leaves were collected in each season to determine preferential sites of bacterial accumulation. The proportion of living and dead bacterial cells could be determined through the use of quantitative real-time PCR in the presence of ethidium monoazide (EMA-qPCR). RESULTS: It was observed a lower bacterial titer at high temperatures in the infected trees relative to titers in mild weather, despite a higher accumulation of the insect vector Diaphorina citri in these conditions. This study also revealed seasonal fluctuations in the titers of bacteria in mature leaves when compared to young leaves. No statistically significant correlation between any meteorological variable, C.Las concentration and D. citri population could be drawn. CONCLUSION: Although, HLB management strategies have focused on vector control, host tree phenology may be important. The evaluation of citrus phenology, C.Las concentration, ACP population and environmental conditions provides insights into the cyclical, seasonal variations of both the HLB pathogen and its vector. These findings should help in the design of integrative HLB control strategies that take into account the accumulation of the pathogen and the presence of its vector.

Identification of putative effector genes and their transcripts in three strains related to 'Candidatus Phytoplasma aurantifolia'.

Molecular mechanisms underlying phytoplasma interactions with host plants are largely unknown. In this study attempts were made to identify effectors of three phytoplasma strains related to 'Ca. P. aurantifolia', crotalaria phyllody (CrP), faba bean phyllody (FBP), and witches' broom disease of lime (WBDL), using information from draft genome of peanut witches' broom phytoplasma. Seven putative effectors were identified in WBDL genome (SAP11, SAP21, Eff64, Eff115, Eff197, Eff211 and EffSAP67), five (SAP11, SAP21, Eff64, Eff99 and Eff197) in CrP and two (SAP11, Eff64) in FBP. No homologs to Eff64, Eff197 and Eff211 in phytoplasmas of other phylogenetic groups were found. SAP11 and Eff64 homologs of 'Ca. P. aurantifolia' strains shared at least 95.9% identity and were detected in the three phytoplasmas, supporting their role within the group. Five of the putative effectors (SAP11, SAP21, Eff64, Eff115, and Eff99) were transcribed from total RNA extracts of periwinkle plants infected with these phytoplasmas. Transcription profiles of selected putative effectors of CrP, FBP and WBDL indicated that SAP11 transcripts were the most abundant in the three phytoplasmas. SAP21 transcript levels were comparable to those of SAP11 for CrP and not measurable for the other phytoplasmas. Eff64 had the lowest transcription level irrespective of sampling date and phytoplasma isolate. Eff115 transcript levels were the highest in WBDL infected plants. This work reports the first sequence information for 14 putative effectors in three strains related to 'Ca. P. aurantifolia', and offers novel insight into the transcription profile of five of them during infection of periwinkle.

In-Depth Transcriptome Sequencing of Mexican Lime Trees Infected with Candidatus Phytoplasma aurantifolia.

Witches' broom disease of acid lime greatly affects the production of Mexican lime in Iran. It is caused by a phytoplasma (Candidatus Phytoplasma aurantifolia). However, the molecular mechanisms that underlie phytoplasma pathogenicity and the mode of interactions with host plants are largely unknown. Here, high-throughput transcriptome sequencing was conducted to explore gene expression signatures associated with phytoplasma infection in Mexican lime trees. We assembled 78,185 unique transcript sequences (unigenes) with an average length of 530 nt. Of these, 41,805 (53.4%) were annotated against the NCBI non-redundant (nr) protein database using a BLASTx search (e-value ≤ 1e-5). When the abundances of unigenes in healthy and infected plants were compared, 2,805 transcripts showed significant differences (false discovery rate ≤ 0.001 and log2 ratio ≥ 1.5). These differentially expressed genes (DEGs) were significantly enriched in 43 KEGG metabolic and regulatory pathways. The up-regulated DEGs were mainly categorized into pathways with possible implication in plant-pathogen interaction, including cell wall biogenesis and degradation, sucrose metabolism, secondary metabolism, hormone biosynthesis and signalling, amino acid and lipid metabolism, while down-regulated DEGs were predominantly enriched in ubiquitin proteolysis and oxidative phosphorylation pathways. Our analysis provides novel insight into the molecular pathways that are deregulated during the host-pathogen interaction in Mexican lime trees infected by phytoplasma. The findings can be valuable for unravelling the molecular mechanisms of plant-phytoplasma interactions and can pave the way for engineering lime trees with resistance to witches' broom disease.

Characterization of in vitro antifungal activities of small and American cranberry (Vaccinium oxycoccos L. and V. macrocarpon Aiton) and lingonberry (Vaccinium vitis-idaea L.) concentrates in sugar reduced fruit spreads.

In this study, cranberry and lingonberry concentrates were added to commercial sugar-reduced fruit spreads (raspberry-Aloe vera, strawberry-guava, and strawberry-lime), and tested for their antifungal activities. Selected strains of the species Absidia glauca, Penicillium brevicompactum, Saccharomyces cerevisiae and Zygosaccharomyces bailii, as well as xerophilic environmental isolates of the genera Penicillium and Eurotium were used for challenge testing. Initially, varying concentrations of synthetic antifungal agents, such as sodium benzoate, potassium sorbate and butyl 4-hydroxybenzoate were tested against these fungi on wort agar containing 31% fructose at different pH values. Subsequently, the experiments were conducted in fruit spreads containing different concentrations of cranberry and lingonberry concentrates. The results of this study demonstrate that these concentrates were able to inhibit growth of visible colonies of xerophilic and non-xerophilic fungi. Cranberry and lingonberry concentrates are interesting candidates for natural preservation against fungal growth in sugar reduced fruit spreads.

Lactose consuming strains of Xanthomonas citri subsp. citri (Xcc) insight into the emergence of natural field resources for xanthan gum production.

Xanthomonas genus possesses a low level of ß-galactosidase gene expression and is therefore unable to produce xanthan gum in lactose-based media. In this study, we report the emergence of some natural field strains of Xanthomonas citri subsp. citri (Xcc) capable to use lactose as a sole carbon source to produce xanthan gum. From 210 Xcc strains isolated from key lime (C. aurantifolia), 27 showed the capacity to grow on lactose containing medium. Xcc lactose consuming strains demonstrated a good level of xanthan production. Amongst all, NIGEBK37 produced the greatest (14.62 g/l) amount of xanthan gum in experimental laboratory conditions. By evaluating the viscosity of the biopolymer at 25 °C, it was demonstrated that xanthan synthesized by strain NIGEBK37 has the highest viscosity (44,170.66 cP). Our results were indicative for the weakness of a commercial strain of Xanthomonas campestris pv. Campestris DSM1706 (Xcc/DSM1706) to produce xanthan in lactose containing medium.

Composição química e atividade antimicrobiana do óleo essencial e anatomia foliar e caulinar de Citrus limettioides Tanaka (Rutaceae) / Chemical Composition and Antimicrobial Activity of Essential Oil and Anatomy of Citrus limettioides Tanaka(Rutaceae) Leaves and Stem

Citrus limettioides (Rutaceae) é utilizada popularmente para tratamento da sinusite, e também como antitérmica e hipotensora. Este trabalho teve como objetivos: identificar os constituintes dos óleos essenciais da folha e da casca dos frutos de C. limettioides; avaliar a atividade antimicrobiana dos óleos essenciais da folha e da casca dos frutos e realizar o estudo anatômico da folha e caule jovem. Os óleos essenciais foram obtidos por hidrodestilação em aparelho de Clevenger modificado e analisados por CG-EM. A concentração inibitória mínima (CIM) foi determinada utilizando o método de microdiluição em caldo frente a bactérias Gram-positivas, Gram-negativas e fungos. Para o estudo anatômico foram realizados cortes histológicos a mão livre e as secções foram coradas com azul de Alcian/safranina, reagente de Steinmetz, lugol e analisados em microscopia de luz. O linalol foi o componente majoritário no óleo essencial das folhas (36,88 a 45,15%) e o limoneno no óleo da casca dos frutos (70,60 a 75,18%). O óleo essencial das folhas apresentou CIM de 2000 μg/mL frente às bactérias e nenhuma atividade frente aos fungos. O óleo essencial da casca dos frutos não apresentou atividade antimicrobiana. A folha é hipoestomática, predominando os estômatos anomocíticos; o mesofilo é dorsiventral com parênquima paliçádico bisseriado e parênquima lacunoso com 11 camadas de células. Cavidades secretoras foram observadas no mesofilo da folha, no pecíolo e no caule, enquanto cristais prismáticos foram identificados em todas as estruturas da folha e do caule. Os dados obtidos no estudo microscópico podem auxiliar na identificação taxonômica desta planta e na avaliação da autenticidade da droga vegetal.

Citrus limettioides (Rutaceae), a sweet lime, is popularly used to treat sinusitis, and as an antipyretic and hypotensive agent.the aim of this study was to identify the constituents of the essential oils from the leaf and fruit peel of C. limettioides, test the antimicrobial activity of these essential oils and perform an anatomical study of the leaf and young stem. Essential oils were isolated by steam distillation in a modified Clevenger apparatus and analyzed by GC-MS. the minimum inhibitory concentrations (MIC) against Gram-positive and Gram-negative bacteria and fungi were determined by broth microdilution.Anatomical studies were performed on freehand histological sections, which were stained with Alcian blue / safranin, Steinmetz reagent and lugol and analyzed by light microscopy. Linalol was the major component in the leaf essential oil (36.88 to 45.15%) and limonene in the fruit peel essential oil (70.60 to 75.18%).the essential oil from leaves showed antimicrobial activity against bacteria (MIC 2000 μg/mL) and no activity against fungi.the essential oil from the fruit peel showed no antimicrobial activity. the leaf is hypostomatic with predominance of anomocytic stomata, the mesophyll is dorsiventral with biseriate palisade and 11 layers of spongy parenchyma cells.Secretory cavities were observed among the mesophyll cells of the leaf, petiole and stem, whilst prismatic crystals were identified in all structures of leaf and stem. the anatomical data obtained in the microscopic study may help in the taxonomic identification of this plant and to verify the authenticity of the drug plant.

Increased resistance against citrus canker mediated by a citrus mitogen-activated protein kinase.

Mitogen-activated protein kinases (MAPK) play crucial roles in plant immunity. We previously identified a citrus MAPK (CsMAPK1) as a differentially expressed protein in response to infection by Xanthomonas aurantifolii, a bacterium that causes citrus canker in Mexican lime but a hypersensitive reaction in sweet oranges. Here, we confirm that, in sweet orange, CsMAPK1 is rapidly and preferentially induced by X. aurantifolii relative to Xanthomonas citri. To investigate the role of CsMAPK1 in citrus canker resistance, we expressed CsMAPK1 in citrus plants under the control of the PR5 gene promoter, which is induced by Xanthomonas infection and wounding. Increased expression of CsMAPK1 correlated with a reduction in canker symptoms and a decrease in bacterial growth. Canker lesions in plants with higher CsMAPK1 levels were smaller and showed fewer signs of epidermal rupture. Transgenic plants also revealed higher transcript levels of defense-related genes and a significant accumulation of hydrogen peroxide in response to wounding or X. citri infection. Accordingly, nontransgenic sweet orange leaves accumulate both CsMAPK1 and hydrogen peroxide in response to X. aurantifolii but not X. citri infection. These data, thus, indicate that CsMAPK1 functions in the citrus canker defense response by inducing defense gene expression and reactive oxygen species accumulation during infection.

Generation and expression in plants of a single-chain variable fragment antibody against the immunodominant membrane protein of Candidatus phytoplasma aurantifolia.

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

Shotgun proteomic analysis of the Mexican lime tree infected with "CandidatusPhytoplasma aurantifolia".

Infection of Mexican lime trees (Citrus aurantifolia L.) with the specialized bacterium "CandidatusPhytoplasma aurantifolia" causes witches' broom disease. Witches' broom disease has the potential to cause significant economic losses throughout western Asia and North Africa. We used label-free quantitative shotgun proteomics to study changes in the proteome of Mexican lime trees in response to infection by "Ca. Phytoplasma aurantifolia". Of 990 proteins present in five replicates of healthy and infected plants, the abundances of 448 proteins changed significantly in response to phytoplasma infection. Of these, 274 proteins were less abundant in infected plants than in healthy plants, and 174 proteins were more abundant in infected plants than in healthy plants. These 448 proteins were involved in stress response, metabolism, growth and development, signal transduction, photosynthesis, cell cycle, and cell wall organization. Our results suggest that proteomic changes in response to infection by phytoplasmas might support phytoplasma nutrition by promoting alterations in the host's sugar metabolism, cell wall biosynthesis, and expression of defense-related proteins. Regulation of defense-related pathways suggests that defense compounds are induced in interactions with susceptible as well as resistant hosts, with the main differences between the two interactions being the speed and intensity of the response.

Proteomic analysis of the Mexican lime tree response to "Candidatus Phytoplasma aurantifolia" infection.

"Candidatus Phytoplasma aurantifolia" is the causative agent of witches' broom disease in the Mexican lime tree (Citrus aurantifolia L.), and is responsible for major tree losses in Southern Iran and Oman. The pathogen is strictly biotrophic, and, therefore, completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We applied a proteomics approach to analyse gene expression in Mexican limes infected with "Ca. Phytoplasma aurantifolia". Leaf samples were collected from healthy and infected plants and were analysed using 2-DE coupled with MS. Among 800 leaf proteins that were detected reproducibly in eight biological replicates of healthy and eight biological replicates of infected plants, 55 showed a significant response to the disease. MS resulted in identification of 39 regulated proteins, which included proteins that were involved in oxidative stress defence, photosynthesis, metabolism, and the stress response. Our results provide the first proteomic view of the molecular basis of the infection process and identify genes that could help inhibit the effects of the pathogen.

Identification of genes differentially expressed during interaction of Mexican lime tree infected with "Candidatus Phytoplasma aurantifolia".

BACKGROUND: "Candidatus Phytoplasma aurantifolia", is the causative agent of witches' broom disease in Mexican lime trees (Citrus aurantifolia L.), and is responsible for major losses of Mexican lime trees in Southern Iran and Oman. The pathogen is strictly biotrophic, and thus is completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. Therefore, we have applied a cDNA- amplified fragment length polymorphism (AFLP) approach to analyze gene expression in Mexican lime trees infected by "Ca. Phytoplasma aurantifolia". RESULTS: We carried out cDNA-AFLP analysis on grafted infected Mexican lime trees of the susceptible cultivar at the representative symptoms stage. Selective amplifications with 43 primer combinations allowed the visualisation of 55 transcript-derived fragments that were expressed differentially between infected and non-infected leaves. We sequenced 51 fragments, 36 of which were identified as lime tree transcripts after homology searching. Of the 36 genes, 70.5% were down-regulated during infection and could be classified into various functional groups. We showed that Mexican lime tree genes that were homologous to known resistance genes tended to be repressed in response to infection. These included the genes for modifier of snc1 and autophagy protein 5. Furthermore, down-regulation of genes involved in metabolism, transcription, transport and cytoskeleton was observed, which included the genes for formin, importin ß 3, transducin, L-asparaginase, glycerophosphoryl diester phosphodiesterase, and RNA polymerase ß. In contrast, genes that encoded a proline-rich protein, ubiquitin-protein ligase, phosphatidyl glycerol specific phospholipase C-like, and serine/threonine-protein kinase were up-regulated during the infection. CONCLUSION: The present study identifies a number of candidate genes that might be involved in the interaction of Mexican lime trees with "Candidatus Phytoplasma aurantifolia". These results should help to elucidate the molecular basis of the infection process and to identify genes that could be targeted to increase plant resistance and inhibit the growth and reproduction of the pathogen.

Identification of Xanthomonas citri ssp. citri host specificity genes in a heterologous expression host.

We provide the first conclusive evidence that Xanthomonas axonopodis pv. citri Asiatic strain (Xac-A) and, in particular, Xac-A(w), a unique citrus canker A strain isolated from Key lime in Wellington, Florida, induces a hypersensitive reaction (HR) in grapefruit leaves. Using the heterologous tomato pathogen X. perforans, as a recipient of the Xac-A(w) genomic library, we identified a 1599-bp open reading frame responsible for HR in grapefruit, but not Key lime, and designated it avrGf1. Xac-A(w)DeltaavrGf1 produced typical, although visibly reduced, citrus canker symptoms (i.e. raised pustules) in grapefruit and typical canker symptoms in Key lime. We also determined that the X. perforans transconjugant carrying an Xac-A(w) hrpG elicited HR in grapefruit and Key lime leaves, and that xopA in X. perforans was partly responsible for HR. Xac-A transconjugants carrying the X. perforans xopA were reduced in ability to grow in grapefruit leaves relative to wild-type Xac-A. The X. perforans xopA appears to be a host-limiting factor. An avrBs3 homologue, which contained 18.5 repeats and induced HR in tomato, was designated avrTaw. This gene, when expressed in a pustule-minus Xac-A(w), did not complement pustule formation; however, pthA(w), a functional pthA homologue, complemented the mutant strain to produce typical pustules in Key lime, but markedly reduced pustules in grapefruit. Both avrBs3 homologues, when expressed in a typical Xac-A strain, resulted in typical citrus canker pustules in grapefruit, indicating that neither homologue suppressed pustule size in grapefruit. Xac-A(w) contains other unidentified factors that suppress development in grapefruit.

Co-occurrence of aflatoxin B(1) and cyclopiazonic acid in sour lime (Citrus aurantifolia Swingle) during post-harvest pathogenesis by Aspergillus flavus.

During hot and humid seasons, extensive rot of sour lime was observed to be caused by Aspergillus flavus. In view of this, investigations were undertaken to obtain data on the production of various toxins by A. flavus during post harvest pathogenesis of sour lime. Sixty percent of the pathogenic A. flavus isolates were detected to be aflatoxin B(1) producers in sour lime tissue. It was also noted that thirty three percent of aflatoxigenic A. flavus isolates had the potential to coproduce cyclopiazonic acid (CPA). Such aflatoxigenic isolates produced quantitatively more CPA (ranging from 250.0 to 2501.3 microg/kg) than aflatoxin B(1) (ranging from 141.3 to 811.7 microg/kg) in the affected sour lime. This study demonstrates for the first time that sour lime are a favourable substrate for aflatoxin B(1) and cyclopiazonic acid production by A. flavus isolates. This is of great concern to the health of consumers.

Nitric oxide: a common antipathogenic factor of plants.

In the present study, nitric oxide synthase/nitric oxide (NOS/NO) status was tested in the host plants infected with fungi, bacteria and virus. In each case cytosolic nitric oxide synthase (Cyt-NOS) of diseased plants was inhibited and inhibition was competitive in nature in respect to l-arginine, the substrate for the enzymic activity. Elevation of host nitric oxide (NO) level before infection using nitric oxide (NO) donor protected disease initiation significantly. The nature of enzyme kinetics and the manner of disease protection by nitric oxide donor (NO-donor) was similar in all the three cases of infection. It was concluded that nitric oxide was a common antipathogenic factor of plants.