Thermal Inactivation Point Research Articles

Uji Infektivitas Tobacco Mosaic Virus (TMV) dan Cucumber Mosaic Virus (CMV) pada Chaenopodium amaranticolor

The infectivity test is one of the completeness in knowing the physical properties of the virus. Determination of the physical properties of the virus can be done by: Thermal inactivation point (TIP), in vitro resistance (Longevity in vitro / LIV), and dilution end point (DEP). The purpose of this study was to determine the infectivity of TMV and CMV in sap through LIV, DEP, and TIP.LIV testing is done by sap incubation until the test time (0 hours, 6 hours, and 24 hours), DEP test is done by diluting sap (not diluted, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7), while TIP test is done by heating sap at room temperature, 50oC, 70oC, 90oC for 10 minutes. The results of this study show TMV is stable compared to CMV.TMV is still infectious in sap storage for 24 hours, while CMV is not infectious between 6-24 hours after incubation. The cutoff point of sap TMV dilution is between 10-5 and 10-6, while CMV has been unable to cause symptoms in indicator plants at dilutions between 10-1 and 10-2. TMV is still infectious at 90oC temperature treatment, while CMV inactivation hot spots are 40oC and 50oC.

Tomato Brown Rugose Fruit Virus: Survival and Disinfection Efficacy on Common Glasshouse Surfaces.

Tomato brown rugose fruit virus (ToBRFV) is a contact-transmitted tobamovirus affecting many tomato growing regions of the world. This study investigated the effects of different glasshouse surfaces on the survival of the virus; the efficacy of different disinfectants; and heat treatment against ToBRFV (surfaces included steel, aluminium, hard plastic, polythene, glass and concrete). A bioassay followed by ELISA was used to check virus viability. ToBRFV survived for at least 7 days on all surfaces tested and on some for at least 6 months. The virus survived for over two hours on hands and gloves. Hand washing was shown to be unreliable for the removal of the virus. Glutaraldehyde and quaternary ammonium compound disinfectants were effective at one hour on all surfaces. Some other disinfectants were effective at one hour of contact time, on all surfaces except concrete. Sodium hypochlorite was partially effective against ToBRFV, even on concrete. A 5 min soak of plastic trays in water at 90 °C was effective at denaturing ToBRFV; however, 5 min at 70 °C was not. Heating infected sap showed the thermal inactivation point to be 90 °C, confirming the hot water treatment results and showing that deactivation was due to the heat treatment and not a washing effect of the water.

Characterization of Diaporthe ampelina isolates and their Sensitivity to Hot-Water Treatments and Fungicides in in vitro

Diaporthe ampelina (=Phomopsis viticola) is one of the most important pathogens causing both cane/leaf spot and wood canker diseases in grape growing countries in the world. In this research, morphological, molecular and pathogenic characterization of 23 D. ampelina isolates were studied and their sensitivity was tested against hot-water treatments and some of the fungicides used in vineyards. Morphologically, the isolates were grouped according to “W type” and “G type” colony appearance and microscopic features. In molecular characterization, beta-tubulin, calmodulin and translation elongation factor (tef1-α) gene regions were amplified with PCR. The nucleotide sequences were analyzed using NCBI-BLAST search and recorded in GenBank, through which species identity was also confirmed. Mycelial viability was tested against hot-water treatments (46 – 50°C for 30 and 45 min) in centrifuge tubes and thermal inactivation point was determined. It was also tested against some of the fungicides (azoxystrobin, boscalid, cyprodinil, tebuconazole, azoxystrobin + cyproconazole + tebuconazole, cyprodinil + fludioxonil, azoxystrobin + tebuconazole and fludioxonil) in vitro and EC50 values were calculated. The morphological and molecular study results showed that all the isolates were D. ampelina and they were pathogenic on wood tissues of vines. Thermal inactivation of “W type” isolates was ensured at 48°C-30 min hot-water treatments. Although this treatment also reduced colony growth of “G type” isolates, it did not inhibit it completely and 48°C-45 min treatment was needed to reach full eradication. Considering fungicide sensitivity, fludioxonil or tebuconazole containing fungicides were the most effective in suppressing the mycelial growth of the fungus. However, azoxystrobin, boscalid, cyprodinil could not perform a strong inhibition when compared to fludioxonil and tebuconazole.

Physical Properties of Potyvirus on Chilli (Capsicum annuum) of Doon Valley in Uttarakhand

A virus is transmitted mechanically in the plant host. Potyviridae is a family of Potyvirus,consisting of a positive-sense RNA genome and filamentous particles. It has a single typeof protein coat. Mostly, this virus is identified in Leguminosae and Solanaceae family. Theplant virus is transmitted by mechanical inoculation and aphids. Chilli, cucurbits, potatoes,peas, tomatoes, and other plants were infected with the bean yellow mosaic virus, pea mosaicvirus, bean yellow mosaic virus, sugarcane mosaic virus, and peanut mosaic virus. Thermalinactivation point, dilution endpoint, longevity in vitro, and longevity in vivo were used toidentify the physical properties of a potyvirus. Several isolates mild and severe were collectedin the chilli plant. The infected plant developed necrotic leaf spots and chlorotic symptoms.

Controlling of multidrug resistant Aeromonas hydrophila infected Nile tilapia (Oreochromis niloticus) using Ah01 and Ah02 virulent bacteriophages isolates

The aim of this work is to investigate the efficiency Ah01 and Ah02 virulent bacteriophages isolated from fish in controlling Aeromonas hydrophila-infected Nile talipa fish. Aeromonas hydrophila strains were resistant to 64% of tested antibiotic, where the antibiotic sensitivity test revealed that among 28 different antibiotics, Aeromonas hydrophila (ATCC 13037) was resistant to 18 antibiotics. In this study, two lytic phages designated as Ah01 and Ah02 specific to Aeromonas hydrophila strain (ATCC 13037) were isolated from fish and agricultural irrigation water, respectively. The isolated phages exhibited tolerance to alkaline and acidic reactions (pH 3–12). The thermal inactivation point was found to be 84 °C for both phages (Ah01 and Ah02). Longevity in vitro of Ah01 and Ah02 phages was estimated to be 21 days at 22 ± 2 °C. The two phage isolates were polyvirulent, since, they were infectious to different species of genus Aeromonas. Transmission electron microscopy analysis revealed that Ah01 and Ah02 phages are of hexagonal head and contractile tail type. Therefore, these two phages belong to family Myoviridae. One step growth curve indicated that the latent periods were15 min for both phages (Ah01 and Ah02). The burst sizes were 102 and 209 PFU/ cell for Ah01 and Ah02 phages, respectively. The RAPD-PCR was used to study the genetic diversity and distances of DNA products of the two virulent phages (Ah01 & Ah02). Six primers with different sizes (P13, N8, B12, A01, S50 and S8) were succeeded to reproduce polymorphic DNA bands. Genetic distances relationship among the two phages were studied based on their similarity indices data of RAPD- PCR analysis. The RAPD primers achieved a rate of polymorphism ranged between 50% and 100%. Complete mortality was recorded in fish injected with Aeromonas hydrophila (ATCC 13037) within two days, but when adding phages, the phage isolates were combined with A. hydrophila strain (ATCC 13037) and intraperitoneal injected in fish, no mortality was detected until the end of experiment (15 days).

Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli.

Bacterial fruit blotch and seedling blight, caused by Acidovorax citrulli, is one of the most destructive diseases of melon and watermelon in many countries. Pathogen-free seed and cultural practices are major pillars of the disease control. However, use of bacteriophages as natural biocontrol agents might also contribute to the disease management. Therefore, we isolated 12 bacteriophages specific to A. citrulli, from phyllosphere and rhizosphere of diseased watermelon plants. The phage strains were characterized based on their host range, plaque and virion morphology, thermal inactivation point, adsorption rate, one step growth curve, restriction fragment length polymorphism (RFLP), and genomic analysis. Transmission electron microscopy of three phage strains indicated that they belong to the order Caudovirales, family Siphoviridae. All phages lysed 30 out of 32 tested A. citrulli strains isolated in Serbia, and did not lyse other less related bacterial species. They produced clear plaques, 2 mm in diameter, on bacterial lawns of different A. citrulli strains after 24 h of incubation. The thermal inactivation point was 66 or 67°C. They were stable at pH 5–9, but were sensitive to chloroform and inactivated in either 5 or 10 min exposure to ultraviolet (UV) light. RFLP analysis using EcoRI, BsmI and BamHI enzymes did not show genetic differences among the tested phages. Adsorption rate and one step growth curve were determined for the Acidovorax phage ACF1. Draft genome sequence of the ACF1 phage was 59.377 bp in size, with guanine-cytosine (GC) content 64.5%, including 89 open reading frames. This phage shared a very high genomic identity with Acidovorax phage ACPWH, isolated in South Korea. Evaluation of systemic nature of ACF1 strain showed that it can be absorbed by roots and translocated to upper parts of watermelon plants where it survived up to 10 days.

DETERMINATION OF PROPERTIES OF A STRAIN OF TOBAMOVIRUS FROM MUCUNA PRURIENS

A virus-infected leaf of Mucuna pruriens was collected and tested for an incidence of a virus through biological properties using various plant host range inoculation tests, longevity-in-vitro, dilution-end-point, thermal-inactivation point and electron microscopy. Results of host range inoculation tests showed the following plant species to be susceptible to the virus; Nicotiana tabacum, N. rustica, N. benthamiana, N. glutinosa, N. occidentalis, N. Sylvesris (Speg and Comes) and others.
 No plant in the family Convolvulacaee, Cucurbitaceae was found to be infected with the virus. However Chenopodium quinoa and C. murale in the family Chenopodiaceae were infected with symptoms of mottling and necrotic lesions. In the family Fabaceae, hybrid cowpea line TVu 76, IT84S – 2114 were infected with mosaic, leaf crinkling and severe leaf curl respectively. The virus had a dilution-end-point of 10-6-10-7, thermal-inactivation-point of 95°C in crude sap of TVu 76. The virus is of agricultural importance because of its incidence on seed coats of legumes which are freely exchanged between agricultural stakeholders.
 
 

Biological and Physico-chemical Properties of Causal Agent of Virus Disease of Coccinia barteri (Hook. f.) Keay in Calabar, Cross River State

The physico-chemical properties of the causal agent of virus disease of Coccinia barteri (Hook. f.) Keay were studied. The virus causing the disease was characterized using diagnostic tools such as host range, longevity in vitro, thermal inactivation point, dilution endpoint and aphid transmission. The virus was mechanically transmitted from the natural host (C. barteri) to the healthy test plants in the green house. In the biological properties, the virus was successfully transmitted by Aphis spiraecola (obtained from Chromolaena odorata (L.) R. M. King & H. Rob.) from infected Cucumeropsis mannii Naudin to a healthy C. mannii in a non-persistent manner and had a narrow host range limited to the family Cucurbitaceae. In the physico-chemical properties based on crude sap with an unknown virus concentration, beyond which infectivity was lost. It was readily inactivated by heating to 35 – 65°C for 10 minutes in determination of thermal inactivation point. The virus had a longevity in vitro of between 4 – 5 days beyond which it was non-infectious. Symptoms induced by the virus were leaf cupping, mottle chlorosis, blisters, stunted growth, rugosity, leaf malformation and mosaic patterns.

ISOLATION OF Aeromonas BACTERIOPHAGE AvF07 FROM FISH AND ITS APPLICATION FOR BIOLOGICAL CONTROL OF MULTIDRUG RESISTANT LOCAL Aeromonas veronii AFs2

Aeromonas isolates from Nile tilapia fish, fish ponds and River water were identified as well as their bacteriophage specific. Also evaluation of antibacterial effect of both nanoparticles and phage therapy against the pathogenic Aeromonas veronii AFs2. Differentiation of Aeromonas spp. was done on the basis of 25 different biochemical tests and confirmed by sequencing of 16s rRNA gene as (A. caviae AFg, A. encheleia AWz, A. molluscorum AFm, A. salmonicida AWh, A. veronii AFs2, A. veronii bv. veronii AFi). All of the six Aeromonas strains were resistant to β-actam (amoxicillin/ lavulanic acid) antibiotics. However, the resistance to other antibiotics was variable. All Aeromonas strains were found to be resistant to ampicillin, cephalexin, cephradine, amoxicillin/clavulanic acid, rifampin and cephalothin. Sensitivity of 6 Aeromonas strains raised against 7 concentrations of chitosan nanoparticles. Using well diffusion method spherically shaped silver nanoparticles AgNPs with an average size of ~ 20 nm, showed a great antimicrobial activity against A. veronii AFs2 and five more strains of Aeromonas spp. At the concentration of 20, 24, 32 and 40 µg/ml. Thermal inactivation point was 84oC for phage AvF07 which was sensitive to storage at 4oC compared with the storage at -20oC. Intraperitoneal injection in fish using phage AvF07 together with A. veronii AFs2, no mortality was shown until the end of experiment (14 days). However, mortality of 43.8% or 50% was obtained after 2 or 3 days, respectively, when chloramphenicol was injected instead of phage.

A68 Characterization and comparative genomic analysis of two Bacillus megaterium lytic bacteriophages

Next-generation sequencing technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. Many Bacillus bacteriophages have been isolated, but very few Bacillus megaterium bacteriophages have been characterized. Here, we describe biological characteristics and whole-genome sequences and their annotations for two new isolates of the B. megaterium bacteriophages (BM5 and BM10), isolated from Egyptian soil samples and representing two different groups according to their host range and amplified fragment length polymorphism profiles. Both phages have been displaying different thermal inactivation points (82 and 78 °C) and pH tolerance range (5–9.2 and 5–8.4 pH) while having the same longevity in vitro (192 h). Electron microscopy observation has proved that both phages belonged to the Myoviridiae family. Furthermore, growth analyses indicated that phages BM5 and BM10 have a shorter latent period (20 and 25 min) and smaller burst size (103 and 117 PFU) than is typical for Bacillus phages. The genome sizes of phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with a modular organization. Bioinformatic analyses of BM5 and BM10 genomes enabled assignments of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of BM5 and BM10 genome structures with other B. megaterium bacteriophages revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7) within L cluster and display different lifestyles (lysogenic and lytic). Sequenced phages encode proteins associated with Bacillus pathogenesis. BM5 does not contain any tRNA sequences, while BM10 genome codes for 17 tRNAs.

PHYSICO-CHEMICAL PROPERTIES OF SOME LISTERIA PHAGES

Listeria monocytogenes is a food borne pathogenic bacteria and caused a dangerous infection of humans. Six lytic bacteriophages specific to L. monocytogenes were isolated from irrigation and sewage waters and named OLG, OLA, OLM, OLD, OLN and OLP. The phages were propagated, and then purified by the ultracentrifugation. Morphological properties of Listeria phages showed that the phages were tailed phages, varied in their sizes and assigned to be a member of Siphoviridae (OLG, OLA, OLM) and Myoviridae (OLN, OLD, OLP) families. All Listeria phages were highly stable under different temperature conditions and its thermal inactivation point was reached to 80°C. The longevity in vitro of the phages was up to 60 days, as well as phages were active at pH values ranging from 4.0 to 12.0. Listeria phages did not lose their infectivity after exposure to UV for 90 min at 35 and 53 cm distances. Results of SDS-PAGE showed that phages had 5-6 protein fragments with molecular weights of 66, 45, 37, 35, 33 and 28 kDa distributed among the six phages.

Biological and molecular evidence of papaya ring spot virus pathotype P from mid-hills of Meghalaya, India

Papaya ring spot virus (PRSV) is the limiting factor of papaya production in India irrespective of agro-climatic conditions and it may cause crop losses up to 85–90%. In this study, diagnostic survey on PRSV was conducted in mid-hills of Ri-Bhoi district, Meghalaya, India during April to June, 2015. Reverse transcription polymerase chain reaction assay detected PRSV infection in 38 of 53 samples collected from surveyed locations with an average incidence of 71.7%. The representative isolate (PRSV-P:Umiam) from mid-hill Meghalaya was maintained in healthy papaya seedlings by standard sap inoculation method and typical symptoms were expressed within 21–24 days post inoculation. Studies on the physical properties revealed that the dilution end point of PRSV-P:Umiam at 10−2, longevity in vitro for 18 h and thermal inactivation point in between 50 and 55 °C. Among the cucurbitaceous hosts, pumpkin (Cucurbita pepo) alone exhibited chlorotic lesions on leaves within 20–25 days post inoculation. The partial coat protein (CP 750 bp) of PRSV-P:Umiam gene shared maximum nucleotide (~ 95%) and amino acid (~ 98%) identity with PRSV isolates reported from northern and eastern India. The CP based phylogeny clustered PRSV-P:Umiam with the PRSV isolates from north and east India.

APPLICATION OF SALMONELLA PHAGE COCKTAIL TO CONTROL SALMONELLA TYPHIMURIUM IN VITRO

Members of the genus Salmonella are among the microbial pathogens associated with dangerous infection. The increased of Salmonella spp. antibiotic resistance has propelled the need of alternate therapeutic methods or strategies. Bacteriophage forms one of these alternate strategies. Six lytic bacteriophages infecting Salmonella Typhimurium were isolated from sewage drain water. Phages were purified biologically by single plaque assay and concentrated using the ultracentrifugation. The phage isolates were named O SM, O SF, O SG, O SP, O SA and O SD. Morphological characters of Salmonella phages showed that all the phage isolates belong to family Myoviridae. All phage isolates were highly stable at room temperature, storage at refrigerator temperature and had thermal inactivation point ranged from 90 to 98 °C. Phages were stable at pH conditions ranging from pH 4 to 12. Phages did not lose their infectivity after exposure to UV for 90 min. at 35 cm and 53 cm distance. The host range of the isolated phages was found that the phages had narrow host range. Phage cocktail with different MOI was used to control Salmonella Typhimurium in vitro. Data revealed that addition of phage cocktail at MOI 10 reduced the Salmonella cells with rate 98.2 % after 4 hrs. from addition. introduction Salmonella spp. belong to family Enterobacteriaceae and widely distributed in nature and often found in the intestinal tract of warm-blooded animals and humans,where they asymptomatically colonize and multiply (Newell & Fearnley, 2003 and Doyle & Erickson, 2006). More than 2,500 serotypes of Salmonella exist and the most prevalent and important Salmonella enterica serotypes reported worldwide are Enteritidis and Typhimurium. These are responsible for 99 % of Salmonellosis in humans and warm-blooded animals. The most common symptoms of Salmonella infection is non-bloody diarrhea and abdominal cramps (Bell and Kyriakides, 2002). Due to foodborne infections by Salmonella are obtained through ingesting contaminated food or water, (Abd El-Aziz, 2013 ) detected high incidence of Salmonella Typhimurium , one of the most frequently isolated serovars from food borne outbreaks throughout the world, in retail raw chicken meat and giblets in Egypt. Bacteriophages are considered an effective weapon against pathogenic bacteria. As a result of development resistant bacteria against antibiotics because of their repeated usage, bacteriophages are used as a safe alternative to control pathogenic bacteria (Abramia et al 2016). In addition, offer a great advantage over antibiotics. First, bacteriophages are specific and target only the pathogens of interest, so the normal gut microflora are not affected. Second, bacteriophages are self-replicating in the bacterial host and lyse bacteria. (Connerton and Connerton, 2005). This paper aims to investigate the incidence of lytic bacteriophages specific for Salmonella in sewage water and study the posibility of their application for controling the Salmonella in vitro.

The Biology and Phylogenetics of Potato virus S Isolates from the Andean Region of South America.

Biological characteristics of 11 Potato virus S (PVS) isolates from three cultivated potato species (Solanum spp.) growing in five Andean countries and 1 from Scotland differed in virulence depending on isolate and host species. Nine isolates infected Chenopodium quinoa systemically but two others and the Scottish isolate remained restricted to inoculated leaves; therefore, they belonged to biologically defined strains PVSA and PVSO, respectively. When nine wild potato species were inoculated, most developed symptomless systemic infection but Solanum megistacrolobum developed systemic hypersensitive resistance (SHR) with one PVSO and two PVSA isolates. Andean potato cultivars developed mostly asymptomatic primary infection but predominantly symptomatic secondary infection. In both wild and cultivated potato plants, PVSA and PVSO elicited similar foliage symptoms. Following graft inoculation, all except two PVSO isolates were detected in partially PVS-resistant cultivar Saco, while clone Snec 66/139-19 developed SHR with two isolates each of PVSA and PVSO. Myzus persicae transmitted all nine PVSA isolates but none of the three PVSO isolates. All 12 isolates were transmitted by plant-to-plant contact. In infective sap, all isolates had thermal inactivation points of 55 to 60°C. Longevities in vitro were 25 to 40 days with six PVSA isolates but less than 21 days for the three PVSO isolates. Dilution end points were 10-3 for two PVSO isolates but 10-4 to 10-6 with the other isolates. Complete new genome sequences were obtained from seven Andean PVS isolates; seven isolates from Africa, Australia, or Europe; and single isolates from S. muricatum and Arracacia xanthorhiza. These 17 new genomes and 23 from GenBank provided 40 unique sequences; however, 5 from Eurasia were recombinants. Phylogenetic analysis of the 35 nonrecombinants revealed three major lineages, two predominantly South American (SA) and evenly branched and one non-SA with a single long basal branch and many distal subdivisions. Using least squares dating and nucleotide sequences, the two nodes of the basal PVS trifurcation were dated at 1079 and 1055 Common Era (CE), the three midphylogeny nodes of the SA lineages at 1352, 1487, and 1537 CE, and the basal node to the non-SA lineage at 1837 CE. The Potato rough dwarf virus/Potato virus P (PVS/PRDV/PVP) cluster was sister to PVS and diverged 5,000 to 7,000 years ago. The non-SA PVS lineage contained 18 of 19 isolates from S. tuberosum subsp. tuberosum but the two SA lineages contained 6 from S. tuberosum subsp. andigena, 4 from S. phureja, 3 from S. tuberosum subsp. tuberosum, and 1 each from S. muricatum, S. curtilobum, and A. xanthorrhiza. This suggests that a potato-infecting proto-PVS/PRDV/PVP emerged in South America at least 5,000 years ago, became endemic, and diverged into a range of local Solanum spp. and other species, and one early lineage spread worldwide in potato. Preventing establishment of the SA lineages is advised for all countries still without them.

Isolation and characterization of basil mosaic virus in Egypt

Different viral infection symptoms were observed on leaves of basil plants (Ocimum basilicum L.), growing at El-Monofia Governorate, Egypt, during 2015–2016. The majority of these symptoms were mosaic and leaf malformation. The pathogenic agent of this disease is known as basil mosaic virus (BsMV). The sensitive hosts to BsMV are Chenopodium amaranticolor and Phaseolus vulgaris cv. (brown beans). The biological properties of BsMV were found to be 10−4, 6 days and 66 °C for dilution end point, longevity in vitro and thermal inactivation point, respectively. The electron micrograph of the isolated virus indicated this virus was found to be isometric particles of approximately, 50 nm in diameter and could be classified as tentative member of the Fabavirus. Real-time PCR detection by the Fab primer pairs was confirmed at Ct value = 25.44 for the total cDNA isolated from infected Basil plants and 27.94 for the cDNA of purified virus particles, while CMV primer pairs showed no amplification for both samples. According to the available reports this seems to be the first record for basil mosaic virus in Egypt.

Biological and Molecular Studies on an Egyptian Potyvirus Isolate from Hyocyamus muticus L.

ANEW isolate of potyvirus was isolated from naturally-infected……. henbane plants (Hyocyamus muticus L.) collected from thefarm of Faculty of Pharmacy, Cairo University in January 2013. Thenaturally infected henbane plants exhibited severe mosaic, rugosity,blistering and malformation. Thermal inactivation point, dilution endpoint and longevity in vitro of virus isolate were found to be 65 oC,10- 4 and 4 days, respectively. Electron micrograph of partial purifiedvirus negat ively s t ained wit h 2% p hosp hot unges t ic acidrevealed the presence of filamentous viruses with size 1440 X14.3nm.Total RNA was extracted from infected henbane plant. Comparativenucleotide sequence analysis for virus showed a high degree ofsimilarity (62 %) with four potyviruses accessions (EU482153: Potatovirus Y isolate Foggia, KF850513: Potato virus Y isolate M3,AM184113: Henbane mosaic virus partial gene for polyprotein-PHYS/H isolate, AY166867: Potato virus Y strain N isolate). Thevirus isolate was published in GenBank with accession numberKM497011.

Biological Control of Sclerotinia sclerotiorum the Causal Agent of Lettuce Rot Disease by Use of Soil Streptomycetes

Sclerotinia sclerotiorum (Lib) De Bary, the causal agent of lettuce rot, is asoil borne fungal pathogen which can cause extensive damage to infected plants or crop and occurs world over. It is difficult to control this pathogen by normal cultural and chemical practices. Our studies have shown that the pathogen can be managed by by use of bacteria belonging to the actinomycetes group. Preliminary screening of 40 soil isolates of actinomycetes from Kerman province of Iran was carried out for testing their ability to inhibit Sclerotinia sclerotiorum by using in vitro agar disk and dual culture evaluations. Among the tested isolates, Actinomycetes isolates B, F and S showed reasonable inhibitory capabilities in dual culture. These isolates were positive forchitinase, Amylase, Protease and Lipase activities but none of them produced HCN. Minimal inhibitory concentration of the active crude extracts varied from1.25 to 0.312 mg/ml and the thermal inactivation point was determined between 90-120°C. All three isolates had fungicidal activity and only isolate F retained its antifungal activity after exposure to chloroform. Significantly, all three isolates reduced the severity of the disease in greenhouse evaluations.

Characterization and Confirmation of Papaya ringspot virus-W Strain Infecting Trichosanthese cucumerina at Tamil Nadu, India

Mosaic and leaf blisters symptoms were observed in Trichosanthese cucumerina (Snake gourd) in regions of coimbatore, India. The biological, physical, serological and molecular properties of the virus isolate were studied. Virus isolate produce chlorotic local lesions on Chenopodium amaranticolor and systemic symptoms on all inoculated cucurbitaceous hosts. The particles appear flexuous rod shape of 750×12 nm under electron microscope. Serologically this isolate identified as strain of PRSV. Dilution end point of virus isolate was 10-4. Thermal inactivation point and Longevity in-vitro of the virus isolate was found to be 55°C and 30 h respectively.

First Report of Lettuce mosaic virus Infecting Lactuca sativa in India.

In January 2012, lettuce (Lactuca sativa L.) plants (19 out of 38) of one of the accessions (EC687345, variety NVRS-10:001818) exhibiting mild mosaic and stunted growth symptoms were observed at the Indian Agricultural Research Institute (IARI) experimental farm, New Delhi. Similar disease symptoms in lettuce plants in India were previously described (3) and the associated virus was characterized for host range, dilution end point, thermal inactivation point, and longevity in vitro. In this study, definitive molecular evidence is presented for the presence of Lettuce mosaic virus (LMV) infecting lettuce in India. Analysis of preparations from leaves of symptomatic samples with an electron microscope revealed flexuous virus particles measuring 750 × 13 nm, suggesting the association of a potyvirus (4). To identify the potyvirus infecting these lettuce plants, the 3' terminal portion of the genome including the part of the nuclear inclusion b (NIb), complete coat protein (CP) region, and 3' untranslated region (UTR) was amplified by RT-PCR, cloned, and sequenced. Total RNA was extracted from infected leaves using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to RT-PCR using potyvirus specific forward (5' ACCACAGGATCCGGBAAYAAYAGYGGDCARCC 3') and reverse (5' CACGGATCCCGGG(T17)V 3') primers (2). PCR products (~1.8 kb) were cloned into pGEM-T Easy vector (Promega, Madison, WI) and sequenced (GenBank Accession No. JQ794776). Sequence comparisons revealed the CP of the virus infecting lettuce (834 bp) shared 96 to 100% nucleotide and deduced amino acid sequence identity with the corresponding regions of LMV isolates AJ306288 and AJ297630 from the United Kingdom, CAA46603 and NC003605 from the United States, AJ278854 and AJ278854 from Brazil, and AJ488153 from China, thus complying with the cut off range of 90 to 99% for identifying isolates/strains of the same virus (1). Similarly, 99 to 100% nucleotide sequence identity was observed with the corresponding region of the 3'UTR (245 bp) while 93 to 96% nucleotide identity of NIb region (654 bp) with LMV isolates. These results confirm that the virus infecting the symptomatic lettuce plants was an isolate of LMV. The amino acid sequences (DAG and WCIEN) conserved among majority of potyviruses were also present. Since the virus is aphid transmissible, its natural infection on other hosts and spread can't be ruled out.

<i>IN VITRO</i> ANTAGONISTIC ANTIFUNGAL-ACTIVITY OF <i>STREPTOMYCES</i> ISOLATE 339 AGAINST <i>MAGNAPORTHE ORYZAE</i>

Biological control offers an environmentally friendly alternative to the use of antimicrobials for controlling plant diseases. A collection of about 100 Actinomycete strains was screened for the ability to produce metabolites that inhibit Magnaporthe oryzae growth in vitro. The Streptomyces isolate 339 showed strong in vitro antagonistic activity against M. oryzae in Agar disc and Well-diffusion methods by producing extracellular antifungal metabolites. The isolate No. 339 was propagated in submerged cultures and active crude was prepared upon which some biological characterization performed. Antifungal activity of this antagonist was of fungistatic type on the pathogen. The active metabolite (s) is polar, soluble in H2O and methanol but insoluble in chloroform, dichloromethane or hexane. Thermal inactivation point of active phase of Streptomyces isolate 339 was 80°C. Antifungal gene from isolate 339 may be a useful candidate for genetic engineering of agriculturally important crop for increased tolerance against M. oryzae.