Leaf Assay Research Articles

Hongos endófitos foliares como candidatos a biocontroladores contra <i>Moniliophthora</i> spp. de <i>Theobroma cacao</i> (Malvaceae) en Ecuador

La adaptabilidad de los hongos endófitos a sus hospedantes, los beneficios ecológicos que le brinda y los diversos mecanismos antagónicos contra plagas que poseen los convierten en una alternativa para el control biológico de enfermedades. Se determinó el potencial de 17 cepas de hongos endofíticos foliares (FEF) obtenidas de tejido sano de Theobroma cacao como candidatas para el control biológico de Moniliophthora roreri (MR) y M. perniciosa (MP). Se evaluaron: i) el micoparasitismo de los FEF frente a colonias de Moniliophthora spp., ii) la acción de los metabolitos crudos de los FEF en el crecimiento, y iii) la habilidad para recolonizar hojas sanas del hospedante mediante ensayos de hojas sueltas. Tres cepas de Lasiodiplodia theobromae fueron las más promisorias: Ec098, Ec151 and Ec157. Estas cepas inhibieron el crecimiento de MR y MP, tanto en el enfrentamiento de las colonias como mediante sus metabolitos y, adicionalmente, recolonizaron el hospedante entre el 80-100 % de las veces. Otras cepas mostraron valores destacados en un indicador, y no deseables en otros. Por ejemplo, la Ec035 (L. theobromae) mostró los niveles más altos de micoparasitismo contra ambos patógenos en la interacción de las colonias, y el segundo mejor por sus metabolitos, pero no pudo reinfectar el hospedante. La cepa Ec059 (Xylaria feejeensis) reinfectó 100 %, pero no mostró los atributos deseados de antagonismo. Por su parte, los metabolitos de Ec107 (Colletotrichum gloeosporioides s.l.) inhibieron a MR en un 60 %, pero también estimularon el crecimiento de MP. Ninguna cepa logró todas las características deseables para un agente de control biológico.

Identification and confirmation of downy mildew (Pseudoperonospora cubensis Berk. & Curt.) resistance sources in cucumber (Cucumis sativus L.)

An experiment was designed to identify the resistance source for downy mildew disease in different genotypes of cucumber. Forty-one cucumber genotypes were screened under natural condition and also under artificial epiphytic condition using detached leaf assay method. Genotype IIHR-438 and Cucumis metuliferus L. showed field resistance with an average PDI of 17.66 and 17.46; AUDPC of 772.24 and 764.48, respectively compared to 73.12 PDI and AUDPC of 3096.64 in highly susceptible genotype of IIHR-389. The disease reaction in selected genotypes of cucumber confirmed by artificial screening was in accordance with disease reaction under natural conditions. Resistant genotype IIHR-438 (14.3 PDI) and C. metuliferus L. (12.8 PDI) had least average PDI as compared to susceptible check Swarna Agethi (58.00 PDI) under artificial condition. Statistical analysis of disease severity data over a period for all the forty-one genotypes using non-linear growth model revealed that 99% variability in disease progression. Screening of genotypes under field conditions, sporulation of pathogen, progress of disease, detached leaf assay and non-linear statistical analysis implied that none of genotypes were found to be immune to downy mildew. Wherein the genotype, IIHR-438 and wild cucumber (C. metuliferus L.) were found resistant to downy mildew disease. Hence, it can be utilized in breeding program to develop resistant cultivar in cucumber against Pseudoperonospora cubensis under tropical conditions of India.

Toxin production by melon root rot fungus, Monosporascus cannonballus

A study was conducted on the possible in vitro production of phytotoxins by the melon root rot pathogen, Monosporascus cannonballus. The culture filtrate of a highly virulent isolate of the fungus was tested for its phytotoxic activity on cucumber by detached leaf assay, electrolyte leakage assay and seedling vigour test. In detached leaf bioassay, necrotic symptoms developed on cucumber leaves 5–7 days after application of the culture filtrate. The culture filtrate of M. cannonballus caused substantial reduction in seed germination, vigor of cucumber seedlings and an increase in electrolyte leakage from leaf tissues compared to the control. Studies on the effect of heat and dialysis on the phytotoxic activity of the culture filtrate revealed that the toxic metabolites are thermostable and have low molecular weight (<14 kDa). To purify the toxin, the culture filtrate was extracted with different solvents which were then tested separately for their phytotoxicity. The results revealed that the water fraction after extraction with chloroform retained the highest phytotoxic activity. The toxin was partially purified by thin layer chromatography (TLC) and the fluorescent compound eluted with an Rf value of 0.6 was analyzed by gas chromatography-mass spectrometry (GC-MS) technique. Mass spectral analysis revealed the presence of two metabolites viz., octocrylene and squalene in the toxin fraction.

A new bacterial blight of pomegranate caused by Pseudomonas sp. in Maharashtra, India

In the past few decades, pomegranate production in India has greatly suffered from a highly catastrophic bacterial blight disease caused by Xanthomonas axonopodis pv. punicae. Various management strategies have failed to control the disease. During surveys carried out in Maharashtra, India in 2010–2015, an unusual yellow pigmented bacterium was consistently associated with pomegranate showing blight symptoms. Based on morphological, biochemical, 16S rRNA and gyrase B gene sequencing, the bacterium was identified as Pseudomonas sp. Pathogenicity was assessed both by a detached leaf assay method (in vitro) and in pomegranate plantlets (in vivo). To the best of our knowledge, we report Pseudomonas sp. as a cause of new bacterial blight disease of pomegranate in Maharashtra, India in addition to Xanthomonas axonopodis pv. punicae.

Development of a protocol to phenotype sweet cherry (Prunus avium L.) for resistance to bacterial canker

Suppression of bacterial canker disease of sweet cherry caused by Pseudomonas syringae pv. syringae (Pss), utilizing resistant scion and rootstock varieties holds promise as a cost-effective management strategy. However, a reproducible and rapid method for screening large breeding populations for resistance to Pss poses a challenge. Sweet cherry cultivars Bing, Sweetheart, Regina, Moreau, Emperor Francis and Rainier were used to examine the effects of Pss isolate, inoculum concentration (1 × 102 to 1 × 108 cfu/ml), leaf age (collected from the tip, middle or base of the shoot) and inoculation assay method (attached versus detached leaf) on disease development. Disease severity was influenced significantly (P ≤ 0.05) by inoculum concentration and the virulence of the Pss isolate. An inoculum concentration of 1 × 108 cfu/ml provided the best disease response in both leaf assays and is recommended for disease screening. Also, a significant Pss isolate x cultivar effect was observed suggesting that proper selection of Pss isolate for disease screening is critical. Disease severity was significantly (P ≤ 0.05) greater for newly expanding leaves in detached assays than for leaves classified as young or old. Disease response among cultivars for attached and detached leaf assays was significantly correlated (r = 0.53, P = 0.002), but the detached leaf assay provided better separation in disease severity among cultivars. We conclude that the genotypic variation in disease response among sweet cherry germplasm can be differentiated based on a detached assay using new leaves, a highly virulent Pss isolate, and an inoculum concentration of 1 × 108 cfu/ml.

Prediction of Sporulation and Germination by the Spider Mite Pathogenic Fungus Neozygites floridana (Neozygitomycetes: Neozygitales: Neozygitaceae) Based on Temperature, Humidity and Time.

Neozygites floridana is a pathogenic fungus and natural enemy of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), which is an important polyphagous plant pest. The aim of this study was to reveal and predict what combination of temperature, relative humidity (RH), and time that enables and promotes primary conidia production and capilliconidia formation in N. floridana (Brazilian isolate ESALQ 1420), in both a detached leaf assay mimicking climatic conditions in the leaf boundary layer and in a semi-field experiment. In the detached leaf assay, a significant number of conidia were produced at 90% RH but the highest total number of primary conidia and proportion of capilliconidia was found at 95 and 100% RH at 25 °C. Positive temperature and RH effects were observed and conidia production was highest in the 8 to 12 h interval. The semi-field experiment showed that for a >90% probability of N. floridana sporulation, a minimum of 6 h with RH >90% and 10 h with temperatures >21 °C, or 6 h with temperatures >21 °C and 15 h with RH >90% was needed. Our study identified suitable conditions for primary- and capilliconidia production in this Brazilian N. floridana isolate. This information provides an important base for building models of a Decision Support System (DSS) where this natural enemy may be used as a tool in Integrated Pest Management (IPM) and a base for developing in vivo production systems of N. floridana.

Biological activity of pod borer, Helicoverpa armigera (Hubner) influenced by chickpea genotypes

Investigations on biological activities of pod borer, Helicoverpa armigera (Hubner) on 20 promising chickpea genotypes using detached leaf assay method at Agriculture Research Station Kalaburagi, UAS, Raichur, Karnataka was carried out during rabi 2016-17. The results revealed that at vegetative and flowering stage there was significantly lesser larval survival (70 to 76.67 %), larval weight (8.37 to 8.90 mg) and damage rate (3.67 to 4.67 of visual Rating in 1-9 Scale) on resistant check ICCL 86111, HC-1 and DBGV-3104 genotypes where as maximum per cent larval survival (86.67 to 90 %), larval weight (13.60 to 14.10 mg) and damage rate (7.67 to 8.00 of visual Rating in 1-9 Scale) was recorded in susceptible checks (ICC-3137, A-1 and JG-11). During pod formation stage, highest weight gain by larvae was noticed on susceptible check ICC-3137 (409.18 mg), on the contrary lowest weight gain by larvae was found on resistant check ICCL-86111 (275.43 mg) also these genotypes recorded more amount of malic acid and trichomes indicating that biological activity of insect was affected through antibiosis mechanism which is one of the component of resistance to H. armigera in chickpea.

Antixenosis and antibiosis mechanisms of resistance to pod borer, Helicoverpa armigera in wild relatives of chickpea, Cicer arietinum

The noctuid pod borer, Helicoverpa armigera is one of the most damaging pests of chickpea, Cicer arietinum. The levels of resistance to H. armigera in the cultivated chickpea are low to moderate, but the wild relatives of chickpea have exhibited high levels of resistance to this pest. To develop insect-resistant cultivars with durable resistance, it is important to understand the contribution of different components of resistance, and therefore, we studied antixenosis and antibiosis mechanisms of resistance to H. armigera in a diverse array of wild relatives of chickpea. The genotypes IG 70012, PI 599046, IG 70022, PI 599066, IG 70006, IG 70018 (C. bijugum), ICC 506EB, ICCL 86111 (cultivated chickpea), IG 72933, IG 72953 (C. reticulatum), IG 69979 (C. cuneatum) and IG 599076 (C. chrossanicum) exhibited non preference for oviposition by the females of H. armigera under multi-choice, dual-choice and no-choice cage conditions. Based on detached leaf assay, the genotypes IG 70012, IG 70022, IG 70018, IG 70006, PI 599046, PI 599066 (C. bijugum), IG 69979 (C. cuneatum), PI 568217, PI 599077 (C. judaicum) and ICCW 17148 (C. microphyllum) suffered significantly lower leaf damage, and lower larval weights indicating high levels of antibiosis than on the cultivated chickpea. Glandular and non-glandular trichomes showed negative correlation with oviposition, while the glandular trichomes showed a significant and negative correlation with leaf damage rating. Density of non-glandular trichomes was negatively correlated with larval survival. High performance liquid chromatography (HPLC) fingerprints of leaf surface exudates showed a negative correlation of oxalic acid with oviposition, but positive correlation with malic acid. Both oxalic acid and malic acid showed a significant negative correlation with larval survival. The wild relatives exhibiting low preference for oviposition and high levels of antibiosis can be used as sources of resistance to increase the levels and diversify the basis of resistance to H. armigera in cultivated chickpea.

Molecular characterization, comparison of screening methods, and evaluation of cross‐pathogenicity of black rot (Xanthomonas campestris pv. campestris) strains from cabbage, choy sum, leafy mustard and pak choi from Taiwan

Choy sum (Brassica rapa var. parachinensis), leafy mustard (Brassica juncea) and pak choi (B. rapa var. chinensis) are highly nutritious components of diets in Taiwan and other Asian countries, and bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a major biotic constraint in these crops. As very little was known about the Xcc strains from these crops in these regions, including their cross‐pathogenicity and aggressiveness on different hosts, Xcc strains were obtained from cabbage (Brassica oleracea var. capitata), choy sum, leafy mustard and pak choi crops in Taiwan. Two previously published PCR‐based assays reliably distinguished the Xcc strains from other Xanthomonas species and subspecies. Phylogenetic analysis based on repetitive sequence‐based PCR assays placed the Xcc strains in a clade distinct from other Xanthomonas species, and also showed host specificity. Although all of the Xcc strains from the different host species were pathogenic on all five Brassica test species in both a detached leaf assay and an intact plant assay, in the intact plant assay they showed differences in virulence or aggression on the different test hosts. The Xcc strains from leafy mustard and pak choi were consistently highly aggressive on all the test host genotypes, but the strains from choy sum and cabbage were less aggressive on leafy mustard and choy sum. The intact plant assay proved more discriminating and reliable than the detached leaf assay for comparing the aggressiveness of Xcc strains on different host genotypes, and so, with the new Xcc strains isolated in this study, will be useful for screening leafy brassica germplasm accessions for resistance to black rot.

An intragenic approach to confer glyphosate resistance in chile (Capsicum annuum) by introducing an in vitro mutagenized chile EPSPS gene encoding for a glyphosate resistant EPSPS protein.

Chile pepper (Capsicum annuum) is an important high valued crop worldwide, and when grown on a large scale has problems with weeds. One important herbicide used is glyphosate. Glyphosate inactivates the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the synthesis of aromatic amino acids. A transgenic approach towards making glyphosate resistant plants, entails introducing copies of a gene encoding for glyphosate-resistant EPSPS enzyme into the plant. The main objective of our work was to use an intragenic approach to confer resistance to glyphosate in chile which would require using only chile genes for transformation including the selectable marker. Tobacco was used as the transgenic system to identify different gene constructs that would allow for the development of the intragenic system for chile, since chile transformation is inefficient. An EPSPS gene was isolated from chile and mutagenized to introduce substitutions that are known to make the encoded enzyme resistant to glyphosate. The promoter for EPSPS gene was isolated from chile and the mutagenized chile EPSPS cDNA was engineered behind both the CaMV35S promoter and the EPSPS promoter. The leaves from the transformants were checked for resistance to glyphosate using a cut leaf assay. In tobacco, though both gene constructs exhibited some degree of resistance to glyphosate, the construct with the CaMV35S promoter was more effective and as such chile was transformed with this gene construct. The chile transformants showed resistance to low concentrations of glyphosate. Furthermore, preliminary studies showed that the mutated EPSPS gene driven by the CaMV35S promoter could be used as a selectable marker for transformation. We have shown that an intragenic approach can be used to confer glyphosate-resistance in chile. However, we need a stronger chile promoter and a mutated chile gene that encodes for a more glyphosate resistant EPSPS protein.

Antifungal Activity of Natural Volatile Organic Compounds against Litchi Downy Blight Pathogen Peronophythora litchii.

Litchi (Litchi chinensis Sonn.) is a commercially important fruit but its production and quality are restricted by litchi downy blight, caused by the oomycete pathogen Peronophythora litchii Chen. Volatile substances produced by a biocontrol antinomycetes Streptomyces fimicarius BWL-H1 could inhibited P. litchii growth and development both in vitro and in detached litchi leaf and fruit infection assay. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) analyses indicated that volatile organic compounds (VOCs) from BWL-H1 resulted in severe damage to the endomembrane system and cell wall of P. litchii cells in vitro and abnormal morphology of appressoria, as well as deformed new hyphae in infection process. VOCs could suppress mycelial growth, sporulation, while with no obvious effect on sporangia germination. Based on gas chromatography-mass spectrophotometric analyses, 32 VOCs were identified from S. fimicarius BWL-H1, the most abundant of which was phenylethyl alcohol. Eight VOCs, including phenylethyl alcohol, ethyl phenylacetate, methyl anthranilate, α-copaene, caryophyllene, humulene, methyl salicylate and 4-ethylphenol, that are commercially available, were purchased and their bioactivity was tested individually. Except for humulene, the other seven tested volatile compounds shown strong inhibitory activity against mycelial growth, sporulation, sporangia germination and germ-tube growth of P. litchii. Especially, 4-ethylphenol showed the highest inhibitory effect on sporulation at a very low concentration of 2 µL/L. Overall, our results provided a better understanding of the mode of action of volatiles from BWL-H1 on P. litchii, and showed that volatiles from BWL-H1 have the potential for control of postharvest litchi downy blight.

Screening Ornamental Cherry (Prunus) Taxa for Resistance to Infection by Blumeriella jaapii

Ornamental flowering cherry trees are important landscape plants in the United States but are susceptible to several serious pests and disease problems. Cherry leaf spot, incited by the fungus Blumeriella jaapii, is characterized by defoliating susceptible trees in late summer, leading to weakening or even death of the tree. To identify resistant plants for use in landscape plantings and in our breeding program, we used a detached leaf assay to screen 69 diverse ornamental flowering cherry taxa for resistance to cherry leaf spot. We found clear differences in susceptibility among the accessions, with seven accessions developing essentially no symptoms at all. A variance decomposition showed that most of the variance (59%) occurred among accessions, indicating that genotype, even more than species, determined susceptibility. The detached leaf assay used in this study is an effective method for screening large numbers of plants for relative resistance to cherry leaf spot. These methods will be particularly useful to characterize germplasm and screen hybrids in breeding and selection programs.

Spatiotemporal distribution of Ascochyta pinodes and Ascochyta pinodella during the winter growing season in France

Ascochyta blight of pea is caused by four related fungi, Ascochyta pisi, Phoma koolunga, Ascochyta pinodes and Ascochyta pinodella. The latter two taxa appear to be much more common and economically significant worldwide but the relative impact of each fungus on ascochyta blight epidemics is not well understood. To study the spatiotemporal distribution of A. pinodes and A. pinodella infecting pea in France, 368 isolates were sampled monthly, from February to May, at three locations (Rennes, Boigneville and Dijon) and molecular markers were used to genotype isolates. The aggressiveness of isolates from the fourth sampling date was estimated using a detached leaf assay on the winter cultivar Enduro. Disease was low during the sampling period as climatic conditions were generally not conducive to disease development (cold temperature, low rainfall). Population genetic analysis showed that 99% of the observed variation could be attributed to variation within populations compared to only 1% among populations. Both species were observed in each location, although A. pinodella was observed at a lower frequency (6–32%). Moreover, results showed that both species could develop on different nodes of the plant. Significant differences in aggressiveness were observed between species and among isolates within species with A. pinodes isolates being significantly more aggressive on average than A. pinodella isolates. These results emphasize the necessity to study the components of disease complexes in order to understand the impact of pathogen species interactions on disease and yield reduction as well as the dynamics of disease epidemics during the cropping season.

Genetic diversity of apple‐ and crabapple‐infecting isolates of Venturia inaequalis in Pennsylvania, the United States, determined by microsatellite markers

SummaryApple scab caused by Venturia inaequalis is the most destructive disease of apple worldwide. In this study, genetic diversity of 101 V. inaequalis isolates from cultivated apples and ornamental crabapples in Pennsylvania (PA, USA) was characterized using 14 microsatellite markers. A total of 157 alleles ranging from 5 (Vitg9/99) to 26 (Vica10/154) per locus were detected. Regardless of the host of origin, isolates were grouped into five clusters, which were largely supported by STRUCTURE and principal coordinate analysis. Cluster analyses based on genetic distances and population structure analysis suggest very small differentiation (PhiPT ranging from 0.016 to 0.103, depending on the population comparison) between apple and crabapple isolates of V. inaequalis. Pairwise comparisons among populations from different locations showed very low differentiation, and POPGENE analysis indicated frequent migration of alleles (Nm = 1.47). In pathogenicity tests using a detached leaf assay, isolates of V. inaequalis from crabapple caused characteristic scab symptoms on apple and were highly virulent. Results of the study indicate that scab lesions in crabapple trees in close vicinity to apple orchards could serve as reservoirs for spread of the pathogen. Movement of inoculum among locations and between hosts may be responsible for the limited population structure observed. Understanding the population structure of V. inaequalis isolates is significant for apple scab management as crabapples are often used as pollinizers and rootstock in apple orchards, and as ornamental trees.

Systemic Colonization and Expression of Disease Symptoms on Bittersweet Nightshade (Solanum dulcamara) Infected with a GFP-Tagged Dickeya solani IPO2222 (IPO2254)

Colonization of Solanum dulcamara (bittersweet nightshade) plants by a GFP-tagged Dickeya solani type strain IPO2222 (IPO2254) was investigated by selective plating and epifluorescence stereomicroscopy (ESM), using in vitro plants and plants grown in compost soil. Replicated experiments were carried out in a growth chamber and the progress of infection and disease symptoms on tissue of the cultured plants, following leaf- and stem-base inoculations with bacteria, was evaluated. Microscopy observations were confirmed by spread-plating dilutions of plant extracts onto agar medium directly after the harvest. In experiments where the stem base of in vitro plants inoculated with a range of inocula of D. solani (104 to 108 colony forming units [cfu] ml-1) was examined at 14 days post infection (dpi), blackleg-like symptoms developed in more than 80% plants together with a reduction of the plant fitness (disease symptoms, weight, height, and appearance). In leaf-inoculated plants at 14 dpi, 15% of the plants exhibited severe blackleg-like symptoms. In detached S. dulcamara leaf assays, IPO2254 survived on the adaxial surface for 14 days at populations of 106 cfu per leaf. Thirty days after stem inoculation of plants grown in compost soil in pots, up to 104 cfu g-1 of GFP-tagged D. solani were found inside the stems. D. solani were detected inside the vascular tissue (xylem vessels) of stems, in the pith tissue in roots, and on the internal surface of the stem hollow. The implications of S. dulcamara infection by D. solani for the long-distance dispersal of the bacterial inoculum are discussed.

In vitro and in vivo Assay for Assessment of the Biological Control Potential of Streptomyces sp. KT

Rhizoctonia solani is fungal plant pathogen that infects many different host plants. Recently, biological control agents that are friendly to the environment and ecosystems have attracted much attention as an alternative to the use of chemical fungicide which have been used worldwide to control soil borne pathogens including R. solani. In this study, 53 strains of actinomycetes isolated from environmental soils, and antifungal activities of them were assessed by the dual culture assay. Strain KT showed strong inhibitory activities against 8 phytopathogenic fungi. A great suppressive effect on R. solani growth was observed in the inoculation test of plants using cucumber and chin-geng-sai. In addition, infection of Bipolaris oryzae also could be suppressed in the detached leaf assay using oats. As a result of genetic analysis, it was shown that KT was a species closely related to Streptomyces lavenduligriseus NRRL B-3173T. However, as far as we know, there is no report for biological control agents using S. lavenduligriseus. This study suggests that the strain KT may useful as biological control agents to suppress various crop diseases.

Stable, fertile somatic hybrids between Sinapis alba and Brassica juncea show resistance to Alternaria brassicae and heat stress

Wild relatives of Brassica are a rich reservoir of genes that are invaluable for the improvement of cultivated species. Sinapis alba is a close relative of crop Brassicas that possesses several desirable traits such as tolerance to Alternaria black spot disease, heat stress, insect pests and nematodes. This study is aimed at developing and characterizing hybrids between Brassica juncea and S. alba with the ultimate goal of transferring genes for tolerance to Alternaria brassicae and heat stress, the traits that are lacking in cultivated Brassica. We generated three hybrids between B. juncea and S. alba through protoplast fusion. The hybridity was confirmed through cytology and molecular markers. While two of the hybrids were symmetric, the third one was asymmetric and had greater resemblance to B. juncea. Hybrids showed some characteristic features of the parents and were fully male and female fertile and also set seeds upon back crossing with the parent species. In vitro leaf assay and field inoculation studies revealed that the hybrids are highly resistant to A. brassicae. Besides, hybrids set seeds at temperature of > 38 °C when parents failed to produce seeds indicating that hybrids possess heat tolerance. These stable hybrids provide a reliable genetic resource for transfer of genes from S. alba into cultivated Brassica species.

Pathogen causing Phalaenopsis soft rot disease - 16S rDNA and virulence characterisation

The pathogen causing Phalaenopsis soft rot disease and developed detached leaf inoculation methods were identified. Based on its 16S rDNA sequences, the pathogen causing soft rot disease in Phalaenopsis was Erwinia chrysanthemi/Dickeya chrysanthemi. Both virulent and avirulent strains were revealed. The detached leaf inoculation assay for E. chrysanthemi/D. chrysanthemi resistance evaluation included wounding and inoculating the detached leaf with 108 CFU/ml of bacteria. Soft rot disease symptoms in the inoculated detached leaf were measurable at 20 h after inoculation. The detached leaf assay was applicable for evaluating Phalaenopsis germplasm and progeny resistance in Phalaenopsis breeding programs.

Characterization and in planta nitrogen fixation of plant growth promoting endophytic diazotrophic Lysinibacillus sphaericus isolated from rice (Oryza sativa)

This study was conducted to isolate and characterize endophytic diazotrophic isolates with multiple plant growth promoting attributes including biocontrol potential from surface sterilized leaf, stem and root samples of various rice cultivars of Kerala. A total of 18 endophytic diazotrophs were isolated using Dobereiner's semisolid N free bromothymol blue media. All the isolates were found to be positive for in vitro acetylene reduction assay (ARA) while only leaf isolate L1 was able to fix nitrogen in planta. Among the 18 nitrogen fixing endophytes isolated, 3 showed in vitro antifungal activities against Rhizoctonia solani, the rice sheath blight pathogen. The in vivo detached leaf assay confirmed the results. All the selected three isolates were proved to produce important plant hormones Indole 3 Acetic acid (IAA), Gibberllic acid (GA) and Cytokinins (Kinetin). The selected isolates augmented the seed germination and seedling vigor. Morphological, physiological, biochemical characteristics and 16SrDNA sequence analysis identified the isolates as Lysinibacillus sphaericus (L1), Klebsiella pneumoniae (S2) and Bacillus cereus (R2). The nif H gene was also amplified to confirm diazotrphy. The isolate L1, the superior endophytic diazotrophic isolate and S2 were also positive for the production of ACC deaminase. This is the first report of Lysinibacillus sphaericus as a nitrogen fixing and plant growth promoting endophyte with biocontrol activity.

Comparative transmission of Bhendi yellow vein mosaic virus by two cryptic species of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae).

The leaf sample from okra plants showing prominent yellow vein mosaic symptoms and healthy plant without any virus symptoms were collected from farmer's field. The presence of begomovirus in the infected sample was confirmed by polymerase chain reaction (PCR) and the amplicons were cloned and sequenced. The genome analysis showed that the isolate in the present study had 99% nucleotide identity with Bhendi yellow vein mosaic virus (BYVMV) revealing it as BYVMV variant. The genetic species of Bemisia tabaci collected from fields were identified as Asia-1 and MEAM-1 genetic species based on silver leaf assay, sequence characterized amplified region marker, and mtCOI gene sequence. The comparative virus-vector relationship of both genetic species of B. tabaci indicates a minimum of two and three B. tabaci in MEAM-1 and Asia-1 genetic species, respectively, per plant were required to transmit the disease. The minimum acquisition access period and inoculation access period of 15 (MEAM-1) and 20min (Asia-1) were required to transmit the YVMD; it was further confirmed by nucleic acid hybridization using coat protein gene-specific probe of BYVMV. With respect to the sex, the female B. tabaci were more efficient in transmitting the disease as compared to male ones in both the genetic species of B. tabaci. The MEAM-1 to transmit the BYVMV more efficiently than Asia-1 genetic species of B. tabaci.