Abstract— In this study, we have used well-known, efficient methods and bioassay for systematic screening of R. solanacearum for identification of its phenotype and biochemical profile, as well as for pathogenicity and virulence. As a result, an aggressive race — Biovar 3 — was most isolated from the potato fields of the Issyk-Kul region, especially in fields where the Picasso variety was grown. The isolated indigenous strains of Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensis strain k1-3 has the potential to be used as a biocontrol agent for the management of the bacterial wilt of potatoes, as indicated by the reduced percentage wilt incidence. Root zone and soil application of Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensis strain k1-3 at a dose of 108 cell/ml significantly reduced disease incidence and increased the growth of potato plants. The disease’s progress was reduced by 60% and 56% in plants inoculated with Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensis strain k1-3, respectively.
Prevalence, occurrence and biochemical characterization of Xanthomonas campes...INNS PUBNET
Xanthomonas campestris pv. vesicatoria the causal organism of bacterial spot in tomato results in heavy losses both in the form of quality and. In this study a survey was carried out to report the incidence of bacterial spot disease of tomato in district Swat. We reported maximum disease incidence in tehsil Kabal (71.66%), followed by Charbagh (61.66%) and Barikot (58.33%). For resistant screening a total of 13 tomato germplasms were screened against the disease. The foliar severity ranged from 3.33% to 73.33%, while severity for fruits was ranged from 18.33% to 30.66%. In case of phenotypic data the highest numbers of fruits obtained were 34, plant height 79.5cm and fruit weight was 470 grams/ten tomatoes. While the lowest average numbers of fruits were 6.67, plant height 45.7cm and fruit weight recorded was 215.67 grams/ten tomatoes. Line 1288 showed highest level of resistance followed by Red-stone. However, line 9708 showed highest susceptibility when exposed to artificial inoculation. Our study showed that bacterial spot is a major issue in some part of Pakistan and germplasm screening are linked to increased host resistance and could offer an important contribution to future integrated bacterial spot management programs.
Laboratory manual for fusarium research 3rd edition Lester BurgessMarcos Buitrago
Authors of this laboratory manual are Lester W. Burgess, Brett A. Summerell, Suzanne Bullock, Kathryn P. Gott and David Backhouse.
Fusarium Research Laboratory
Department of Crop Sciences
University of Sydney
August, 1994
in vitro screening of larvicidal and insecticidal activity of methanolic extr...IJEAB
The aim of this work was to evaluate the larvicidal and insecticidal activity of the selected plants namelyArtocarpus altilis, Piper betle and Artocarpus heterophyllus. The leaves of Artocarpus altilis and Artocarpus heterophyllus and roots of Piper betle were subjected to methanolic solvent extraction for the isolation of various bioactive constituents. The evaluation of larvicidal activity was carried out using late third instar larvae of Drosophila melanogaster. The insecticidal activity of extracts was studied against adult Bruchus pisorum, Tribolium castaneum, Sitophilus oryzae and was evaluated by direct contact application method .Nucleic acids and protein contents are regarded as important biomarkers of the metabolic potential of cells, as these play the main role in regulating the different activities of cells. Piper betle and Artocarpus heterophyllusextractshad a reducing effect on the nucleic acid and protein content in the larvae in a dose dependent manner whereas Artocarpus altilis extract did not exhibit any significant larvicidal activity. Piper betle and Artocarpus heterophyllusextractsshowed good insecticidal activity whereas A.altilis extract showed poor insecticidal activity. The results of the present study clearly indicate that Piper betle and Artocarpus heterophyllus extracts can be developed as ecofriendly larvicides and were also quite effective as insecticides for providing a better and excellent alternate for the control of insects.
Prevalence, occurrence and biochemical characterization of Xanthomonas campes...INNS PUBNET
Xanthomonas campestris pv. vesicatoria the causal organism of bacterial spot in tomato results in heavy losses both in the form of quality and. In this study a survey was carried out to report the incidence of bacterial spot disease of tomato in district Swat. We reported maximum disease incidence in tehsil Kabal (71.66%), followed by Charbagh (61.66%) and Barikot (58.33%). For resistant screening a total of 13 tomato germplasms were screened against the disease. The foliar severity ranged from 3.33% to 73.33%, while severity for fruits was ranged from 18.33% to 30.66%. In case of phenotypic data the highest numbers of fruits obtained were 34, plant height 79.5cm and fruit weight was 470 grams/ten tomatoes. While the lowest average numbers of fruits were 6.67, plant height 45.7cm and fruit weight recorded was 215.67 grams/ten tomatoes. Line 1288 showed highest level of resistance followed by Red-stone. However, line 9708 showed highest susceptibility when exposed to artificial inoculation. Our study showed that bacterial spot is a major issue in some part of Pakistan and germplasm screening are linked to increased host resistance and could offer an important contribution to future integrated bacterial spot management programs.
Laboratory manual for fusarium research 3rd edition Lester BurgessMarcos Buitrago
Authors of this laboratory manual are Lester W. Burgess, Brett A. Summerell, Suzanne Bullock, Kathryn P. Gott and David Backhouse.
Fusarium Research Laboratory
Department of Crop Sciences
University of Sydney
August, 1994
in vitro screening of larvicidal and insecticidal activity of methanolic extr...IJEAB
The aim of this work was to evaluate the larvicidal and insecticidal activity of the selected plants namelyArtocarpus altilis, Piper betle and Artocarpus heterophyllus. The leaves of Artocarpus altilis and Artocarpus heterophyllus and roots of Piper betle were subjected to methanolic solvent extraction for the isolation of various bioactive constituents. The evaluation of larvicidal activity was carried out using late third instar larvae of Drosophila melanogaster. The insecticidal activity of extracts was studied against adult Bruchus pisorum, Tribolium castaneum, Sitophilus oryzae and was evaluated by direct contact application method .Nucleic acids and protein contents are regarded as important biomarkers of the metabolic potential of cells, as these play the main role in regulating the different activities of cells. Piper betle and Artocarpus heterophyllusextractshad a reducing effect on the nucleic acid and protein content in the larvae in a dose dependent manner whereas Artocarpus altilis extract did not exhibit any significant larvicidal activity. Piper betle and Artocarpus heterophyllusextractsshowed good insecticidal activity whereas A.altilis extract showed poor insecticidal activity. The results of the present study clearly indicate that Piper betle and Artocarpus heterophyllus extracts can be developed as ecofriendly larvicides and were also quite effective as insecticides for providing a better and excellent alternate for the control of insects.
CHARACTERIZATION OF STREPTOMYCES SCABIES ISOLATESijabjournal
Potato, (Solanum tuberosum L,) have various biotic constraints in its production due to pest attack. Among these, common scab caused by streptomyces scabies in an important disease in potato which causes economic loss with respect to plant yield and quality of tubers. The present study was conducted to determine the pathogenicity, pathogenic variation, characterization of morphological, physiological and
biochemical aspects of Streptomyces specie associated with potato tubers grown in Rawalpindi district.Severity data and pathogenic variation of disease was studied by using different isolation and characterization techniques. Isolation and characterization of Streptomyces spp. From potato tubers will
guide the researchers about the causative strains of common scab of potato present in the particular area.
In vitro evaluation of Trichoderma viride and Trichoderma harzianum for its e...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Effect of time and proportion of leaf harvest on pest, forage and root yields...Agriculture Journal IJOEAR
Dearth of knowledge exists regarding the leaf harvest intensity and frequency thresholds that support optimum forage and fresh storage root yields in Sierra Leone. A study was carried out to assess the effects of leaf harvesting time and proportion on Cylas puncticollis infestation, growth and yield of sweet potato in the inland valley swamp and upland ecologies of Njala. Treatment combinations comprised of two varieties ("Kabia" and "Gbanie"), four leaf harvest regimes: 0, 30 60 and 90 days after planting (DAP); and four-leaf harvest intensities (0, 25, 50 and 100%). The experiment was laid out in a randomized complete block design (RCBD) with three replications. Data collected included Cylas puncticollis severity on vines and storage roots, root dimensions and numbers, fresh foliage and storage root yields. The results revealed that leaf harvesting twice at 25 and 50% contributed more to optimum forage and storage root yields and related attributes of sweet potatoes compared to other treatments. The present study suggests that good agronomic management of sweet potato that supports optimum forage and storage root yields should be selected to meet the dual purpose for which it is grown. These findings serve as good guide for incorporation of leaf harvesting time, proportion of leaf harvest in germplasm assessment and new population development objectives.
Mass Production of Paecilomyces Lilacinus by using Different Cultivation Medi...Agriculture Journal IJOEAR
Paecilomyces lilacinus is a common saprophytic, filamentous fungus. Morphological characters of Paecilomyces lilacinus were separate mycelium, hyaline, conidia white to pink colored and formation of phialides. The growth of Paecilomyces lilacinus carried out on SDA media at room temperature was better than incubator. Various solid substrates like Rice, Wheat bran, and Sorghum were evaluated for the mass multiplication of fungus Paecilomyces lilacinus. Added dextrose and antibiotics in solid media for mass multiplication at room temperature. Among all the substrate Wheat bran recorded the maximum spore count of 7. 1 10-8 spore/ml followed by Sorghum 5. 4 10-8 spore/ml and Rice 5. 1 10-8 spore/ml after 20 days. Also dry mycelia weight or biomass of fungus Paecilomyces lilacinus without an incubator was more than using an incubator.
Successful strategies against bacterial wilt in SSAHarahagazwe
This presentation was made at the Refresher Course on Potato Seed Production organized by Wageningen UR at the Kenya Plant Health Inspectorate Service (KEPHIS) in Nairobi, Kenya on the 25th November to the 6th December 2013.
CHARACTERIZATION OF STREPTOMYCES SCABIES ISOLATESijabjournal
Potato, (Solanum tuberosum L,) have various biotic constraints in its production due to pest attack. Among these, common scab caused by streptomyces scabies in an important disease in potato which causes economic loss with respect to plant yield and quality of tubers. The present study was conducted to determine the pathogenicity, pathogenic variation, characterization of morphological, physiological and
biochemical aspects of Streptomyces specie associated with potato tubers grown in Rawalpindi district.Severity data and pathogenic variation of disease was studied by using different isolation and characterization techniques. Isolation and characterization of Streptomyces spp. From potato tubers will
guide the researchers about the causative strains of common scab of potato present in the particular area.
In vitro evaluation of Trichoderma viride and Trichoderma harzianum for its e...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Effect of time and proportion of leaf harvest on pest, forage and root yields...Agriculture Journal IJOEAR
Dearth of knowledge exists regarding the leaf harvest intensity and frequency thresholds that support optimum forage and fresh storage root yields in Sierra Leone. A study was carried out to assess the effects of leaf harvesting time and proportion on Cylas puncticollis infestation, growth and yield of sweet potato in the inland valley swamp and upland ecologies of Njala. Treatment combinations comprised of two varieties ("Kabia" and "Gbanie"), four leaf harvest regimes: 0, 30 60 and 90 days after planting (DAP); and four-leaf harvest intensities (0, 25, 50 and 100%). The experiment was laid out in a randomized complete block design (RCBD) with three replications. Data collected included Cylas puncticollis severity on vines and storage roots, root dimensions and numbers, fresh foliage and storage root yields. The results revealed that leaf harvesting twice at 25 and 50% contributed more to optimum forage and storage root yields and related attributes of sweet potatoes compared to other treatments. The present study suggests that good agronomic management of sweet potato that supports optimum forage and storage root yields should be selected to meet the dual purpose for which it is grown. These findings serve as good guide for incorporation of leaf harvesting time, proportion of leaf harvest in germplasm assessment and new population development objectives.
Mass Production of Paecilomyces Lilacinus by using Different Cultivation Medi...Agriculture Journal IJOEAR
Paecilomyces lilacinus is a common saprophytic, filamentous fungus. Morphological characters of Paecilomyces lilacinus were separate mycelium, hyaline, conidia white to pink colored and formation of phialides. The growth of Paecilomyces lilacinus carried out on SDA media at room temperature was better than incubator. Various solid substrates like Rice, Wheat bran, and Sorghum were evaluated for the mass multiplication of fungus Paecilomyces lilacinus. Added dextrose and antibiotics in solid media for mass multiplication at room temperature. Among all the substrate Wheat bran recorded the maximum spore count of 7. 1 10-8 spore/ml followed by Sorghum 5. 4 10-8 spore/ml and Rice 5. 1 10-8 spore/ml after 20 days. Also dry mycelia weight or biomass of fungus Paecilomyces lilacinus without an incubator was more than using an incubator.
Successful strategies against bacterial wilt in SSAHarahagazwe
This presentation was made at the Refresher Course on Potato Seed Production organized by Wageningen UR at the Kenya Plant Health Inspectorate Service (KEPHIS) in Nairobi, Kenya on the 25th November to the 6th December 2013.
This presentation is done by 2010/2011 batch of Export Agriculture students of Uva Wellassa University of Sri Lanka as a requirement for the subject which is “Fruit & Vegetable Cultivation”. Note that the information included here is relevant to Sri Lankan condition.
Isolation, identification of antagonistic rhizobacterial strains obtained fro...Shazia Shahzaman
Plant growth promoting rhizobacteria (PGPR), are associated with roots, found in the rhizosphere and can directly or indirectly enhance the plant growth. In this study soil was collected from rhizosphere of chickpea fields of different areas of Rawalpindi division of Pakistan. PGPR were isolated, screened and characterized. Eight isolates of rhizobacteria (RHA, RPG, RFJ, RC, RTR, RT and RK) were isolated from Rawalpindi division and were characterized. The antagonistic activity of these PGPR isolates against root infecting fungi (Fusarium oxysporum and Verticillium spp.,) was done and production of indole acetic acid (IAA), siderophore and P-solubilization was evaluated. The isolates RHA, RPG, RFJ, RC, RRD and RT were found to be positive in producing siderophore, IAA and P-solubilization. Furthermore, most of the isolates showed antifungal activity against Fusarium oxysporum, and Verticillium spp. The rhizobacterial isolates RHA, RPG, RFJ, RC, RRD, RTR, RT and RK were used as bio-inoculants that might be beneficial for chickpea cultivation as the rhizobacterial isolates possessed the plant growth promoting characters i.e. siderophore, IAA production, phosphate solubilization. In in vitro tests, Pseudomonas sp. and Bacillus spp. inhibited the mycelial growth of the fungal root pathogens. The isolates (RHA and RPG) also significantly increased (60-70%) seed germination, shoot length, root length of the chickpea. The incidence of fungi was reduced by the colonization of RHA and RPG which enhanced the seedling vigor index and seed germination. The observations revealed that isolates RHA and RPG is quite effective to reduce the fungal root infection in greenhouse, and also increases seed yields significantly. These rhizobacterial isolates appear to be efficient yield increasing as well as effective biocontrol agent against fungal root pathogen.
MICROORGANISMS ASSOCIATED WITH THE SPOILAGE OF CUCUMBER, GARDEN EGG AND PAWPA...paperpublications3
Abstract: A total of nine cucumbers, each of Garden egg and pawpaw samples were collected from Wurukum, High level and Wadata markets and cultured on appriopate agar, for colony count and isolation of bacteria according to their cultural and biochemical characteristics. The results revealed that garden egg from High Level Market had the highest bacterial count (1.9x105cfu/g) and the least was pawpaw from High Level Market (1.1 x 105 cfu/g). However, it was not statistically significant. The bacteria isolated were; Propianol bacteria (23.3%), Escherichia coli (16.6%), Staphylococcus aureus (36.7%), Bacillus spp (10.0%) and Corynebacteria (13.3%). The fungal isolates were Aspergillus flavus (10%), Aspergillus fumigatus (20%), Aspergillus nidulus (10%), Aspergillus terreus (20%) and mucor (40%). The result of this study shows fruits sold in Wurukum, High Level Market and Wadata Market are contaminated and may cause harm to consumers, so measures such as proper handling should be taken to control the contamination of these fruits.
MOLECULAR ANALYSIS OF BACTERIAL GENE CODING CHITINASE ENZYMES, FAMILY 19 STR...IJSIT Editor
Fungal phytopathogens pose serious problems worldwide in the cultivation of economically
important plants.
Chemical fungicides are extensively used in current agriculture.However, excessive use of chemical
fungicides in agriculture has led to deteriorating human health , environmental pollution, damaged to
ecosystem and development of pathogen resistance to fungicide.
Because of the worsening problems in fungal disease control , a serious search is needed to identify
alternative methods for plant protection, which are less dependent on chemicals and are more
environmentally friendly. Microbial antagonists are widely used for the biocontrol of fungal plant diseases.
Many species of actinomycates, particulary those belonging to the genus sterptomyces, are well known as
antifungal biocontrol agents that inhibit several plant pathogenic fungi.
Another way biological control has been developed as an alternative of chemicals to tock with plant
pathogenic fungi. Considering high presence of chitin in fungal cell wall, chitinase enzyme is camped as an
effective biocontrol agent against phytopathogenic fungi. Streptomyces bacteria are able to produce various chitinase enzymes, chitinases produced by streptomyces belong to the families 18 and 19 glycosyl hydrolases.
The antifungal activity is mostly shown by fomily 19 Chitinases. In comparison with bacterial family 18
chitinases, the specific hydrolyzing activity of chitinase 19 against soluble and in soluble chitinous substrates
has been markedly higher. Considering the importance of family to investigate antifungal potential of
streptomyces bacteria isolated from east Azarbijan region soils based on molecular identification of family 19
chitinase. encoding gene in these bacteria.
To aim the purpose 110 soil samples were collected from East Azarbaijan and 310 strepomyces
isolates were selected using macroscopic and microscopic observations. DNA genomic of all of the isolates
were extracted and PCR reactions was done using chitinase 19 designed primers as marker.
Totally isolates were selected with molecular selection and antagonistic test were done. One of the isolates
exhibit the most strong antifungal activity.
The strain was identified using 16srDNA gene, and the chitinase encoding gene were amplified partially to
prove the PCR selection. Finally the bacterium were introduced as potentially biological fertilizer.
The objectives of this study to investigate the occurrence and etiology of tomato malformation recently observed in Egypt and illustrate the responsibility of associated phytoplasma. Also, the study was extended to characterize associated phytoplasma, based on molecular techniques.
The effects of explant rotation, medium types, JA and GA3 additions on in vit...Agriculture Journal IJOEAR
— This study investigates the effects on the in vitro microtuber formations of Solanum tuberosum L. cv. Marfona species, effected by the direction of planting the explants (horizontal or vertical), the type of medium (solid or two-phase), adding Jasmonic acid (JA) (0.0, 10 ng/L, 1 µg/L and 0.2 mg/L) and Gibberellic acid (GA 3) (0.0 and 0.2 mg/L). The cultures were incubated in a climate chamber at 22-25 o C, and were subjected to a light intensity of 145 µmol m-2 s-1 for 8 hours in light and 16 hours in dark photoperiods (short day) for 4-6 weeks. Microtuber production was inhibited when GA 3 was added. The maximum number of microtubers was observed when the explants were planted vertically and were grown in two-phase medium which did not contain GA 3 and had 10 ng/L JA present. It was determined that two-phase medium with 0.2 mg/L JA but without GA 3 was the most favourable medium for tuber growth (for both height and width). The best microtuber formation on single node explants were observed to occur in the short day photoperiod (8 hours light/16 hours dark) in a two-phase medium that contained 0.2 mg/L JA without the addition of GA 3. The results shows that the effect caused by JA works antagonistically with that of GA 3 thus causing the resulting microtuber formation observed.
The objective of this study was to examine the antiviral activity of native lactoferrin against Potato virus x, the most important virus that severely affects potato crop and productivity in Egypt, using tissue culture technique and spraying the plants in greenhouse by the aqueous solution of lactoferrin.
Antibiotic Enteric Resistant Bacteria are Abundant on Lettuce from Urban Agri...YogeshIJTSRD
The presence of antibiotic resistant bacteria on lettuce from wastewater irrigated fileds in the town of Yaounde was investigated. Lettuce samples were collected from three agricultural fields located in three districts Ekounou, Ezazou and Nkolbisson , over a period of four months. Resistance to amoxicillin, tetracycline, ciprofloxacin, sulfamethoxazole trimethoprim and kanamycin were determined. Isolated bacteria presented highest resistance to amoxicillin 95.8 and lowest resistance to ciprofloxacin 89.2 . The resistance rate of bacteria was the highest in March 93.1 , and lowest in May 89.9 . Antibiotic resistant bacteria identified were belonged to the genus Salmonella, Bacillus, Staphylococcus, Shigella, klebsiella, Pseudomonas, citrobacter, Micrococcus, Enterococcus, Lactobacillus, Escherichia, and Streptococcus. Bacillus cereus was the most abundant 17.5 . The identified isolates were all multidrug resistant, except Lactobacillus which was only resistant to amoxicillin. The obtained results demonstrate the health risk posed by the cultivation and consumption of lettuce from urban agriculture in the city of Yaounde, which could constitute a strong vector for antibiotic resistance dissemination. Brunelle Tchakounte Betbui | Merveille Tamema Masse | Blaise Pascal Bougnom "Antibiotic Enteric Resistant Bacteria are Abundant on Lettuce from Urban Agriculture in the City of Yaounde" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-3 , April 2021, URL: https://www.ijtsrd.com/papers/ijtsrd39971.pdf Paper URL: https://www.ijtsrd.com/biological-science/microbiology/39971/antibiotic-enteric-resistant-bacteria-are-abundant-on-lettuce-from-urban-agriculture-in-the-city-of-yaounde/brunelle-tchakounte-betbui
Mutation Induction for Improvement of Banana (Musa Spp). "Berangan Cv. Intan-...paperpublications3
Abstract: In vitro mutation induction by using gamma ray at 20, 30, 40 and 60Gy was used to generate variability in triploid banana "Berangan cv. Intan (AAA), so as to provide the opportunity to select plants with desirable characters such as early fruiting and short stature. Mutation frequency increased with increased dosage whereas, survival and capacity to regenerate decreased with increased doses. Time to initiation varied from 4-8 weeks for gamma-irradiated materials compared to 2-3 weeks in the control. It appeared that the higher the dose, the longer it took for shoot initiation. The exposure of shoot-tip meristem pieces to radiation doses produced wide variation in growth and morphogenetic performance. Mutagenic treatments induced 2 to 3- fold increases in variability in both quantitative and qualitative traits at different stages, in vitro; at nursery and field. For the field-grown plants; the proportion varied from 2.9% for the control plants to 16.8% for 60 Gy and 20.1% for 40 Gy-treatments, while for treatments at 20 and 30 Gy variations was at 18% and 19.2%, respectively. The frequency of variants was highest in 40 Gy followed by 30 Gy and 20 Gy, while it was very low in 60 Gy except for plant stature (dwarfism or stunted growth). Earliness to flowering variants were recorded at 20, 30 and 40 Gy at low frequencies (0.6%, 0.7% and 1.7%) respectively, while none was observed for 60 Gy treatments. As in vitro mutation induction could create genetic variability as well as many undesirable variants, it is highly desirable to integrate in vitro mutation with a selection system that can screen for large mutagen treated population. The useful variants recorded for earliness to flowering were selected for 30 and 40 Gy treatments. 40 Gy showed high frequency in earliness as compared with 20 and 30 Gy., which came to flowering as early as 6 – 6.5 months compared to 7-8 months for control. The useful dwarf, which considered as desirable traits, showed a balance of height and girth.
Mutation Induction for Improvement of Banana (Musa Spp.) Berangan Cv. Intan-AAApaperpublications3
Abstract: In vitro mutation induction by using gamma ray at 20, 30, 40 and 60Gy was used to generate variability in triploid banana "Berangan cv. Intan (AAA), so as to provide the opportunity to select plants with desirable characters such as early fruiting and short stature. Mutation frequency increased with increased dosage whereas, survival and capacity to regenerate decreased with increased doses. Time to initiation varied from 4-8 weeks for gamma-irradiated materials compared to 2-3 weeks in the control. It appeared that the higher the dose, the longer it took for shoot initiation. The exposure of shoot-tip meristem pieces to radiation doses produced wide variation in growth and morphogenetic performance. Mutagenic treatments induced 2 to 3- fold increases in variability in both quantitative and qualitative traits at different stages, in vitro; at nursery and field. For the field-grown plants; the proportion varied from 2.9% for the control plants to 16.8% for 60 Gy and 20.1% for 40 Gy-treatments, while for treatments at 20 and 30 Gy variations was at 18% and 19.2%, respectively. The frequency of variants was highest in 40 Gy followed by 30 Gy and 20 Gy, while it was very low in 60 Gy except for plant stature (dwarfism or stunted growth). Earliness to flowering variants were recorded at 20, 30 and 40 Gy at low frequencies (0.6%, 0.7% and 1.7%) respectively, while none was observed for 60 Gy treatments. As in vitro mutation induction could create genetic variability as well as many undesirable variants, it is highly desirable to integrate in vitro mutation with a selection system that can screen for large mutagen treated population. The useful variants recorded for earliness to flowering were selected for 30 and 40 Gy treatments. 40 Gy showed high frequency in earliness as compared with 20 and 30 Gy., which came to flowering as early as 6 – 6.5 months compared to 7-8 months for control. The useful dwarf, which considered as desirable traits, showed a balance of height and girth.
— The microbiological content of Lettuce (a vegetable), commonly vended in the Benin metropolis of Edo state were evaluated. Five vending locations were chosen for the study. Whole and soft rot samples were purchased and analysed for microbiological composition. Results showed high counts in soft rot samples in lettuce. Nutrient agar plated lettuce samples had bacterial counts in the range of 2.0x 103 to 4.7x10 7. Pseudomonas species was the dominant species found in lettuce samples. Bacillus species was isolated from one location in the lettuce samples. Mac Conkey agar plated lettuce plated had bacterial counts in the range of 2.3 x 10 3 to 5.7x 10 7. Enterobacter species, E. coli, and Klebsiella species were the dominant species isolated. Though, Proteus species was isolated from lettuce samples obtained from location five only. The study observes that consuming soft rot samples could pose a risk of introducing pathogens to the consumer due to their high microbial counts and could be detrimental to the health of the consumer.
Pseudomonas fluorescens as plant growth promoting Rhizo- Bacteria and biologi...Innspub Net
The use of plant growth promoting rhizobacteria (PGPR) to control disastrous diseases in many crops has been considered important recently. The research was conducted to evaluate several bacterial strains to control white rust in chrysanthemum. The research consisted of two chronological experiments, in vitro and in vivo testing of bacterial isolate against the disease. 16 bacteria isolates were collected, purified and applied on the rust-infected leaf. Three isolates showed more effective in suppressing white rust during in vitro testing and further identification confirmed these strains, Pf Kr 2, Pf Smd 2 and Pf Ktl were grouped into P. flourescens. In vivo testing of the Pf isolates also revealed consistent performances of these three Pf isolates in retarding the growth of fungal Puccinia horiana and even more effective than Azotobacter sp. and Azospirilium sp. The production of ethylene on the leaf was coincidence with the slower development and lower disease intensity on the treated plants. Among the three strains, Pf Kr 2 showed stronger suppression to the disease. Further investigations are needed to further elucidate the existence of specific interrelation between Pf strains and plant genotypes or cultivars. Prior to a selection of good bacterial inoculants, it is recommended to select cultivars that benefit from association with these bacteria. Get the full articles at: http://www.innspub.net/ijaar/pseudomonas-fluorescens-as-plant-growth-promoting-rhizo-bacteria-and-biological-control-agents-for-white-rust-disease-in-chrysanthemum/
Epidemiology, etiology and management of fusarium wilt of muskmelonNageshb11
Fusarium wilt of muskmelon
Similar to Identification of Ralstonia Solanacearum in Kyrgyzstan’s Potato Fields and the Possibility of Using Biocontrol Agents Against this Pathogen (20)
Micro RNA genes and their likely influence in rice (Oryza sativa L.) dynamic ...Open Access Research Paper
Micro RNAs (miRNAs) are small non-coding RNAs molecules having approximately 18-25 nucleotides, they are present in both plants and animals genomes. MiRNAs have diverse spatial expression patterns and regulate various developmental metabolisms, stress responses and other physiological processes. The dynamic gene expression playing major roles in phenotypic differences in organisms are believed to be controlled by miRNAs. Mutations in regions of regulatory factors, such as miRNA genes or transcription factors (TF) necessitated by dynamic environmental factors or pathogen infections, have tremendous effects on structure and expression of genes. The resultant novel gene products presents potential explanations for constant evolving desirable traits that have long been bred using conventional means, biotechnology or genetic engineering. Rice grain quality, yield, disease tolerance, climate-resilience and palatability properties are not exceptional to miRN Asmutations effects. There are new insights courtesy of high-throughput sequencing and improved proteomic techniques that organisms’ complexity and adaptations are highly contributed by miRNAs containing regulatory networks. This article aims to expound on how rice miRNAs could be driving evolution of traits and highlight the latest miRNA research progress. Moreover, the review accentuates miRNAs grey areas to be addressed and gives recommendations for further studies.
Artificial Reefs by Kuddle Life Foundation - May 2024punit537210
Situated in Pondicherry, India, Kuddle Life Foundation is a charitable, non-profit and non-governmental organization (NGO) dedicated to improving the living standards of coastal communities and simultaneously placing a strong emphasis on the protection of marine ecosystems.
One of the key areas we work in is Artificial Reefs. This presentation captures our journey so far and our learnings. We hope you get as excited about marine conservation and artificial reefs as we are.
Please visit our website: https://kuddlelife.org
Our Instagram channel:
@kuddlelifefoundation
Our Linkedin Page:
https://www.linkedin.com/company/kuddlelifefoundation/
and write to us if you have any questions:
info@kuddlelife.org
Characterization and the Kinetics of drying at the drying oven and with micro...Open Access Research Paper
The objective of this work is to contribute to valorization de Nephelium lappaceum by the characterization of kinetics of drying of seeds of Nephelium lappaceum. The seeds were dehydrated until a constant mass respectively in a drying oven and a microwawe oven. The temperatures and the powers of drying are respectively: 50, 60 and 70°C and 140, 280 and 420 W. The results show that the curves of drying of seeds of Nephelium lappaceum do not present a phase of constant kinetics. The coefficients of diffusion vary between 2.09.10-8 to 2.98. 10-8m-2/s in the interval of 50°C at 70°C and between 4.83×10-07 at 9.04×10-07 m-8/s for the powers going of 140 W with 420 W the relation between Arrhenius and a value of energy of activation of 16.49 kJ. mol-1 expressed the effect of the temperature on effective diffusivity.
UNDERSTANDING WHAT GREEN WASHING IS!.pdfJulietMogola
Many companies today use green washing to lure the public into thinking they are conserving the environment but in real sense they are doing more harm. There have been such several cases from very big companies here in Kenya and also globally. This ranges from various sectors from manufacturing and goes to consumer products. Educating people on greenwashing will enable people to make better choices based on their analysis and not on what they see on marketing sites.
"Understanding the Carbon Cycle: Processes, Human Impacts, and Strategies for...MMariSelvam4
The carbon cycle is a critical component of Earth's environmental system, governing the movement and transformation of carbon through various reservoirs, including the atmosphere, oceans, soil, and living organisms. This complex cycle involves several key processes such as photosynthesis, respiration, decomposition, and carbon sequestration, each contributing to the regulation of carbon levels on the planet.
Human activities, particularly fossil fuel combustion and deforestation, have significantly altered the natural carbon cycle, leading to increased atmospheric carbon dioxide concentrations and driving climate change. Understanding the intricacies of the carbon cycle is essential for assessing the impacts of these changes and developing effective mitigation strategies.
By studying the carbon cycle, scientists can identify carbon sources and sinks, measure carbon fluxes, and predict future trends. This knowledge is crucial for crafting policies aimed at reducing carbon emissions, enhancing carbon storage, and promoting sustainable practices. The carbon cycle's interplay with climate systems, ecosystems, and human activities underscores its importance in maintaining a stable and healthy planet.
In-depth exploration of the carbon cycle reveals the delicate balance required to sustain life and the urgent need to address anthropogenic influences. Through research, education, and policy, we can work towards restoring equilibrium in the carbon cycle and ensuring a sustainable future for generations to come.
Willie Nelson Net Worth: A Journey Through Music, Movies, and Business Venturesgreendigital
Willie Nelson is a name that resonates within the world of music and entertainment. Known for his unique voice, and masterful guitar skills. and an extraordinary career spanning several decades. Nelson has become a legend in the country music scene. But, his influence extends far beyond the realm of music. with ventures in acting, writing, activism, and business. This comprehensive article delves into Willie Nelson net worth. exploring the various facets of his career that have contributed to his large fortune.
Follow us on: Pinterest
Introduction
Willie Nelson net worth is a testament to his enduring influence and success in many fields. Born on April 29, 1933, in Abbott, Texas. Nelson's journey from a humble beginning to becoming one of the most iconic figures in American music is nothing short of inspirational. His net worth, which estimated to be around $25 million as of 2024. reflects a career that is as diverse as it is prolific.
Early Life and Musical Beginnings
Humble Origins
Willie Hugh Nelson was born during the Great Depression. a time of significant economic hardship in the United States. Raised by his grandparents. Nelson found solace and inspiration in music from an early age. His grandmother taught him to play the guitar. setting the stage for what would become an illustrious career.
First Steps in Music
Nelson's initial foray into the music industry was fraught with challenges. He moved to Nashville, Tennessee, to pursue his dreams, but success did not come . Working as a songwriter, Nelson penned hits for other artists. which helped him gain a foothold in the competitive music scene. His songwriting skills contributed to his early earnings. laying the foundation for his net worth.
Rise to Stardom
Breakthrough Albums
The 1970s marked a turning point in Willie Nelson's career. His albums "Shotgun Willie" (1973), "Red Headed Stranger" (1975). and "Stardust" (1978) received critical acclaim and commercial success. These albums not only solidified his position in the country music genre. but also introduced his music to a broader audience. The success of these albums played a crucial role in boosting Willie Nelson net worth.
Iconic Songs
Willie Nelson net worth is also attributed to his extensive catalog of hit songs. Tracks like "Blue Eyes Crying in the Rain," "On the Road Again," and "Always on My Mind" have become timeless classics. These songs have not only earned Nelson large royalties but have also ensured his continued relevance in the music industry.
Acting and Film Career
Hollywood Ventures
In addition to his music career, Willie Nelson has also made a mark in Hollywood. His distinctive personality and on-screen presence have landed him roles in several films and television shows. Notable appearances include roles in "The Electric Horseman" (1979), "Honeysuckle Rose" (1980), and "Barbarosa" (1982). These acting gigs have added a significant amount to Willie Nelson net worth.
Television Appearances
Nelson's char
Natural farming @ Dr. Siddhartha S. Jena.pptxsidjena70
A brief about organic farming/ Natural farming/ Zero budget natural farming/ Subash Palekar Natural farming which keeps us and environment safe and healthy. Next gen Agricultural practices of chemical free farming.
Diabetes is a rapidly and serious health problem in Pakistan. This chronic condition is associated with serious long-term complications, including higher risk of heart disease and stroke. Aggressive treatment of hypertension and hyperlipideamia can result in a substantial reduction in cardiovascular events in patients with diabetes 1. Consequently pharmacist-led diabetes cardiovascular risk (DCVR) clinics have been established in both primary and secondary care sites in NHS Lothian during the past five years. An audit of the pharmaceutical care delivery at the clinics was conducted in order to evaluate practice and to standardize the pharmacists’ documentation of outcomes. Pharmaceutical care issues (PCI) and patient details were collected both prospectively and retrospectively from three DCVR clinics. The PCI`s were categorized according to a triangularised system consisting of multiple categories. These were ‘checks’, ‘changes’ (‘change in drug therapy process’ and ‘change in drug therapy’), ‘drug therapy problems’ and ‘quality assurance descriptors’ (‘timer perspective’ and ‘degree of change’). A verified medication assessment tool (MAT) for patients with chronic cardiovascular disease was applied to the patients from one of the clinics. The tool was used to quantify PCI`s and pharmacist actions that were centered on implementing or enforcing clinical guideline standards. A database was developed to be used as an assessment tool and to standardize the documentation of achievement of outcomes. Feedback on the audit of the pharmaceutical care delivery and the database was received from the DCVR clinic pharmacist at a focus group meeting.
Identification of Ralstonia Solanacearum in Kyrgyzstan’s Potato Fields and the Possibility of Using Biocontrol Agents Against this Pathogen
1. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 146
Identification of Ralstonia Solanacearum in Kyrgyzstan’s Potato
Fields and the Possibility of Using Biocontrol Agents Against this
Pathogen
Tinatin Doolotkeldieva1
, Saykal Bobusheva2
Kyrgyz-Turkish Manas University, Faculty of Agriculture, Plant Protection Department, Bishkek, Kyrgyzstan
Abstract— In this study, we have used well-known, efficient methods and bioassay for systematic screening of R.
solanacearum for identification of its phenotype and biochemical profile, as well as for pathogenicity and virulence. As a
result, an aggressive race — Biovar 3 — was most isolated from the potato fields of the Issyk-Kul region, especially in fields
where the Picasso variety was grown. The isolated indigenous strains of Streptomyces diastatochromogenesstrain sk-6 and
Streptomyces bambergiensis strain k1-3 has the potential to be used as a biocontrol agent for the management of the
bacterial wilt of potatoes, as indicated by the reduced percentage wilt incidence. Root zone and soil application of
Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensis strain k1-3 at a dose of 108
cell/ml
significantly reduced disease incidence and increased the growth of potato plants. The disease’s progress was reduced by
60% and 56% in plants inoculated with Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensis
strain k1-3, respectively.
Keywords— bacterial wilt of potatoes, biocontrol, R. solanacearum,Streptomyces diastatochromogenes, Streptomyces
bambergiensis.
I. INTRODUCTION
Ralstonia solanacearum is a soil-borne pathogen that naturally infects roots. It exhibits a strong and tissue-specific tropism
within the host, specifically invading and highly multiplying in the xylem vessels (Smith, 1896, Yabuuchi et al., 1995). It
causes a wilt disease in more than 450 plant species of 54 botanical families across the globe (Allenet al., 2005, Agrios,
1997, Stevenson, 2001). Ralstonia solanacearum has been studied intensively both biochemically and genetically, and has
long been recognized as a model system for the analysis of pathogenicity (Staskawicz et al., 2001). It is well adapted to life
in soil in the absence of host plants (Granada and Sequeira, 1983), thereby providing a good system to investigate functions
governing adaptation to such an ecological niche. Considering the genetic diversity among the strains responsible for wilting
disease in different plants, the pathogen is now termed as Ralstonia solanacearum species complex (Genin and Denny,
2012). In a traditional way, this pathogen has been classified into five races with respect to their host specificity and six
biovars according to their biochemical properties (French et al., 1995).
The first signs of the disease are shown in the beginning of the flowering and tuber formation. Plants suddenly wilt; the
leaves turn yellow, shrivel, and droop. The lower basal part of the stem softens and rots. A typical feature of brown rot is the
splitting of the stems; the crosscut of them follows a drop of bacterial exudates. Subsequently, the bacteria penetrate into the
stolon, then into young tubers, causing browning of the vascular ring. From sections of the affected vessels and tubers
follows brown mucus (Daughtrey, 2003). Bacterial wilt occurs mainly in tropic, sub-tropic and warm temperature zones
(Jaunet and Wang, 1999). However, this disease has spread to more temperate areas (Kim et al., 2003).
Ralstonia solanacearum is a b-Proteobacterium, whose complete genome sequence was presented by analysis of strain
GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb
megaplasmid. The genome encodes many proteins potentially associated with a role in pathogenicity (Salanoubat et al.,
2002).
As the disease is widely distributed, it has a wide host range and is mainly soil-borne; it is difficult to control with chemicals
and cultural practices (Grimaultet al., 1993). Biological control strategies may either help development of alternative
management measures or be integrated with other practices for effective disease management at the field level. Several
microorganisms have been tried out with variable success for biological control of bacterial wilt (Shekhawat et al., 1993).
Effective microorganisms (EM) are a mixture of beneficial microorganisms, which can increase the crop yield and also
protect against plant pathogens (Higa, 1999, Lwin and Ranamukhaarachch, 2006). It is a mixed culture of photosynthetic
bacteria, Azotobacter, Streptomyces, and Lactobacillus spp., which improve crop yield by increasing photosynthesis, nitrogen
fixation, controlling soil diseases, and accelerating decomposition of lignin material in the soil (Hussain et al., 1993).
2. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 147
Biocontrol of bacterial wilt by plant growth-promoting rhizobacteria provided disease control and increased yields in
greenhouses (Guoet al.,2004, Lemessa and Zeller, 2007). Research on microbial antagonists, such as Candida ethanolica has
shown promise for bacterial wilt control (Lwin and Ranamukhaarachch, 2006). Toyota and Kimura (1996) reported the
suppressive effect of some antagonistic bacteria on R. solanacearum. Three antagonists isolated from soil (Bacillus
megaterium, Enterobacter cloacae, Pichia guillermondii) and Candida ethanolica showed high potential for disease
suppression and also increased fruit weight, biomass, and plant height (Nguyen and Ranamukhaarachchi,2010).
In Kyrgyzstan, the potato (Solanum tuberosum) is a staple product for the population.Recently, farms in different regions
have started to grow the varieties such as Picasso, Sante, and Nevskiy, which were imported from Russia and other countries
of the world, besides local potato varieties. Approximately 32% of potato yields are lost every year due to viral, bacterial,
fungal, and pest attacks to potato tubers and potato plants [An overview of the emergence and spread of major pests and
diseases, 2011). Brown, slimy bacterial bacterios of potatoes (bacterial wilt, or wilt) caused by Ralstonia solanacearum
potatoes (RS) is a relatively a new disease in the fields of Kyrgyzstan. There are still no data and records of the scientists and
experts on the biology and distribution of this disease in the potato crop regions of Kyrgyzstan. There are suggestions that
this bacterial disease was brought with imported planting material to Kyrgyzstan from neighboring countries. So, the disease
has been found in Russia in 1999 by a quarantine inspection only in an area of 0.06 hectares, planted with the imported
variety Sante, then the infestation of potato was found in many regions of Russia: in the Urals, Far East, and Western and
Eastern Siberia (http://www.kartofel.org/bolezn/bacteria/bacwilt.htm). There is a particular threat to potato production
(especially seed production) because of asymptomatic cases of these bacterial diseases, as apparently healthy tubers have a
margin hidden (latent) infection and pose a threat to crops the following year. This makes it important to be able to identify
the disease in the contaminated material. Still, the prevalence and host range of races and biovars of Ralstonia solanacearum
is unknown in the potato-cultivated regions of Kyrgyzstan, but it is becoming increasingly clear that this species causes
disease in the vegetation period and in storage after harvesting. Biological control of Ralstonia solanacearum is still in its
research phase in Kyrgyzstan. The objective of this study was to distinguish the biovars of Ralstonia solanacearum by using
biochemical and ELISA tests, PCR analysis, to determine the prevalence of pathogen races in commercial potato fields of
Kyrgyzstan and to develop the biocontrol agents to reduce the harmfulness of latent infection of seed tubers.
II. MATERIAL AND METHODS
2.1 Origin of isolates
For direct isolation of Ralstonia solanacearum, the potato tubers of Picasso, Sante, and Nevskiy varieties were used, which
were collected in the fall 2012 and 2013 from Issuk-Kul and Chy regions of country. All isolates from potato fields came
from individual tubers of different plants. Each tuber was placed in an individual plastic bag after harvest.
2.2 Cultural characterization
The infected part of tubers was cut using a sterile, sharp knife. A suspension from plant ooze and exudates was prepared in
sterile distilled water and then streaked onto Kelman’s tetrazolium chloride (TZC) agar and 2% sucrose peptone agar (SPA).
After incubation at 28°C for 24 to 36 hours, chartered colonies of Ralstonia solanacearum were selected on mediums.
Isolates of R. solanacearum were maintained in sterile distilled water for the identification steps and stored at room
temperature. Pure cultures were tested by biochemical and enzyme-linked immunosorbent assay (ELISA) methods. A
mobility, gram negative reaction, catalase, amylolytic and lecithinase activity, liquefaction of gelatin, saccharolytic enzymes,
the formation of indole, and other biochemical properties, were determined. For pigment formation tests, the liquid mediums
of meat-peptone broth and tryptophan broth were used. More consistent results were obtained when L-tyrosine was added to
the medium. Denitrification ability was tested using the semi-solid medium: 10% peptone, 5% NaCL, 2.0% KNO3, 3.0 %
Bacto Agar, and Hiss reagent.
2.3 The biovars test
The pathogen species is subdivided into races based on host range. To identify the biovar of pathogen species we have used a
biochemical method based on the utilization of the disaccharides cellobiose, lactose, and maltose, and oxidation of the
hexose alcohols dulcitol, mannitol, and sorbitol (French et al.,1995, Staskawicz et al., 2001).
2.4 Accumulation of Ralstonia solanacearum isolates in the host-plant tissue
Healthy potato tubers of different varieties were used for accumulation the pathogen culture in the host cell. The tubers were
washed thoroughly with water, and then were sterilized in 96% ethyl alcohol. After that, they were thoroughly rinsed in
3. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 148
sterile water, cut into pieces, and placed in Petri dishes on wet, sterile filter paper. Bacterial suspensions at a concentration of
108
CFU/ml were infiltrated into potato slices. Inoculated slices were incubated at the optimal temperature (28°C) for the
bacteria. The optimum moisture ensured the rapid growth of bacteria.
2.5 Pathogenesis assays on potato seedlings and plants
Three potato (Solanum tuberosum) cultivars were used for pathogenicity tests: Picasso (highly sensitive), Sante (medium
resistance) and Nevskiy (high resistance).Three-week-old plants grown in soil were inoculated by soil drench without root
severing. The concentration of bacterial inoculums was 108
CFU/ml. The experiment was repeated at least twice, giving a
total of six test plants. Inoculated plants were kept in a room with natural light and a mean temperature of 25–27°C. The
percentage of plants showing wilting symptoms was recorded during 28 days.
2.6 Immunoblot ELISA test (Agdia).
The Ralstonia solanacearum (RS) ELISA test was used with plant samples exhibiting symptoms of Rs and with bacterial
culture samples. According to the protocol of DAS ELISA of Agdia, the samples were added to a microplate coated with
monoclonal antibodies to EPS of Rs. If EPS is present in the sample, it is bound by antibodies and captured on the microplate
during the incubation period. After incubation, the plate was washed to remove the unbound sample. An enzyme conjugate
solution, containing a monoclonal antibody conjugated to peroxidase, is added and bound to any captured EPS. After
incubation the plate is washed to remove any unbound conjugate. This final binding creates a sandwich of the target analyst
between the two specific antibodies. Wells in which a blue color developed indicated positive results. Wells in which there
was no significant color development indicated a negative result. Test results were valid only if positive control wells gave a
positive result and buffer wells remained colorless.
2.7 Characteristics of biocontrol agents
The antagonistic microorganisms: Streptomyces species and Trichoderma lignorium used in this study were obtained from
the laboratory collection of Phytopathology Laboratory (Plant Protection Department, Faculty of Agriculture, Kyrgyz-
Turkish Manas University, Kyrgyzstan). Streptomyces diastatochromogenesstrain sk-6, Streptomyces bambergiensis strain
sk-2-2 andS. cirratus SK 2-8 were isolated from the rhizosphere of wild plants in the elevated mountain ecosystem of Son-
Kul place (3400 m above sea level).The 16S rRNA genes of these strains were PCR amplified with 27f and 1522r primers,
and PKS genes were screened by polyketide synthetase primers. Trichoderma lignorium was isolated from soil where red
beets were grown. They had been selected as active antagonistic organisms after successive screenings against gram positive
and gram negative bacteria, as well as pathogen fungi.
2.8 Evaluation of Streptomyces species and Trichodermalignorium as potential antagonists against the pathogen
Ralstonia solanacearum in vitro
For screening the antagonists for activity against Ralstonia solanacearum we have used several in vitro methods: the cross-
culture method, the filter paper method, and the perforated agar plate method. In the cross-culture method, each antagonist
was streaked across the Petri plates, and after 72 hours, R. solanacearum was applied as streaks perpendicular to the
antagonist. Plates were incubated at 28°C for 24–48 hours, after which interactions were examined and the distance of the
inhibition zones was measured and recorded. In the filter paper method, the pathogen was poured onto Petri plates and the
antagonist was introduced as filter paper disks 5 mm in diameter. The filter paper disks were dried at 40°C for one hour, and
then placed on cultured pathogen plates. Plates were incubated for 24–48 hours at 28°C before interactions were examined.
In the perforated agar plate method (also known as the bore-hole method) the pathogen was poured as a thin layer onto the
plate, and then an 8 mm hole was made in the agar in which the antagonists were introduced. Plates were incubated for 24–
48 hours at 30°C before examining antagonist-pathogen interactions.
2.9 Effects of antagonistic strains on potato wilt
Soil and potato seedlings were treated with the antagonists and their effects studied in indoor conditions. Microbial
suspensions of antagonists and R. solanacearum were adjusted to a concentration of 108
CFU/ml by measurement at a
wavelength of 585 nm (UV/VIS Spectrophotometer, JENWAY, UK, 2011).
The Picasso potato (highly sensitive) cultivar was used for evaluation of antagonistic actinomycetes strains Streptomyces
diastatochromogenesstrain sk-6 and Streptomyces bambergiensis strain k1-3 in vivo tests. A sufficient volume of soil
autoclaved at 121°C for 30 minutes was used to fill pots 18 cm tall and 30×13 cm in diameter, to a height of 15 cm. Picasso
4. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 149
cultivar healthy potato tubers were planted in soil. Three-week-old seedlings were watered with 20 ml (108
cell/ml) of
pathogen suspension/per plant every day. Then the wilt-susceptible seedlings were watered every day with 20 ml (108
cell/ml)
of antagonist suspension over seven days. In control pots, the wilt-susceptible seedlings were watered with 20ml plain water.
The experiment was repeated at least twice, giving a total of six test plants in each variant. Inoculated plants were kept in a
room with natural light and a mean temperature of 28°C. The percentage of plants showing the wilting symptom was
recorded during 28 days. Disease record: The percentage wilt incidence (PWI) was calculated as follows:
2.10 Statistical analysis
Data were analyzed following GLIM program of Royal Society of London (Crawley, 1995). Significant differences between
two mean values due to treatments or varieties and their interaction at a crop growth stage were computed by comparing their
significant levels at P<0.05.
III. RESULTS
3.1 Origin of isolates and organism characteristics
We analyzed potato tubers of Picasso, Sante, and Nevskiy varieties. Ralstonia solanacearum — as a pathogen of bacterial
wilt — was obtained from the Picasso variety. Twelve isolates from the potato fields of Issuk-Kul and seven isolates from
Chy regions were identified as Ralstonia solanacearum species.
Large, elevated, fluidal, and white colonies of isolated bacteria were grown after two days on the TZC medium, and white,
fluidal with whorls characteristic colonies appeared on SPA. The organism was capable of growing at 28–36°C temperatures
aerobically and it does not form endospores. The bacteria take the form of slightly thick sticks with dimensions of 0.7–0.9
microns, it is gram negative, motile, and non-encapsulated. Cells of obtained isolates Ralstonia solanacearum were motile
when viewed microscopically, indicating its ability to be virulent. The isolates were catalase and oxidase positive. New
isolates of Ralstonia solanacearum were able to reduce nitrate to nitrite. Changing the medium color to red and the formation
of a layer of foam from an intensive gas release indicates a complete reduction of nitrate and denitrification (Fig.1).
FIG.1. FORMATION OF A LAYER OF FOAM FROM AN INTENSIVE GAS RELEASE, INDICATING A COMPLETE
REDUCTION OF NITRATE AND DENITRIFICATION BY RALSTONIA SOLANACEARUM
3.2 The biovars test
Specific host range and distribution of Ralstonia solanacearum depends on the race and the biovars of the pathogen. In Table
1, the data related to the relationship of race, biovars, host range, and geographic distribution of Ralstonia solanacearum are
summarized. It is known the five races of potato brown rot. The most dangerous is Race 3, which affects the potatoes at low
temperatures. The infection persists for a long time in plant debris and potato tubers (in a latent form), and it is common in
temperate regions. Its main sources are infected soil, crop residues, and weeds of the genus Solanaceae (Kim et al., 2003).
5. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 150
TABLE 1
RACES AND BIOVARS OF RALSTONIA SOLANACEARUM. (ADAPTED FROM DAUGHTREY, 2003)
Race Host Range Geographic Distribution Biovar
1 Wide Asia, Australia, the Americas 3,4
1
2 Banana, other Musa spp. Caribbean, Brazil, Worldwide 1
3 Potato, some other Solanaceae,
Geranium;
a few other species
Worldwide, except US and Canada 2
4 Ginger Asia 3,4
5 Mulberry China 5
Isolated races of Ralstonia solanacearum by biochemical characteristics were classified as a 3-biotype, so they were able to
oxidize the disaccharides cellobiose, lactose, and maltose and the hexose alcohols dulcitol, mannitol, and sorbitol. Table 2
illustrates the classification into biovars based on this method. When bromomethylBlau was used as an indicator the medium
becomes yellow as a result of oxidation, and when Andred indicator was used the medium changes to red. Transformation of
this substrate by isolates occurred slowly, for example as shown in Fig. 3 in the presence of bromomethylBlau indicating that
an oxidation of dulcitol occurred only after 12 days.
TABLE 2
CLASSIFICATION OF RALSTONIA SOLANACEARUM INTO BIOVARS. (ADAPTED FROM FRENCH ET AL, 1995)
Physiological Tests Biovars
1 2 3 4 5
Utilization of disaccharides
Cellobiose ─ + + ─ +
Lactose
─
+ +
─
+
Maltose
─
+ +
─
+
Oxidation of alcohols
Dulcitol
─ ─ + + ─
Mannitol
─ ─ + + ─
Sorbitol
─ ─ + + ─
2 days 4-5 days 7-9 days 10-12 days
FIG. 3. AN OXIDATION OF DULCITOL BY RALSTONIA SOLANACEARUM ISOLATES IN THE PRESENCE OF
BROMOMETHYLBLAU INDICATOR
6. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 151
3.3 Accumulation of Ralstonia solanacearum isolates in the host-plant tissue
In many cases, Ralstonia solanacearum bacteria are closely interrelated with secondary pathogens such as the causative
agent of soft rot Erwinia carotovora var. atroseptica(Fegan and Prior,2005).This creates some difficulties for the isolation of
a pure culture of Ralstonia solanacearum from the affected tissue. For the accumulation of the culture of the pathogen in the
host cell and to determinate its virulence, bacterial suspensions of Ralstonia solanacearum at a concentration of 108
CFU/ml
were infiltrated into sterile healthy potato slices. They were incubated at lower temperatures (22°C), in a moisture chamber.
The optimum moisture level ensured the rapid growth of bacteria. The organism quickly began to multiply in infected host
cells. On the third day, dark ringed circles appeared on potato slices. Gradually, a rotting of the entire surface of potato slices
started. In five days, there was a complete decay, with the release of odors and the tissues turning into mucous (Fig. 4). Of all
the varieties tested, only Picasso showed high sensitivity to rotting at low temperatures. These results allowed us to identify
which varieties are more resistant or more susceptible to this disease. It is important to provide advice to farmers on which
varieties are the best to grow in different climatic zones of the Republic. This test has additionally confirmed that obtained
Ralstonia solanacearum new isolates are belong to biovar 3, which can survive at low temperatures. Some researchers have
noted in their results that high temperatures and high soil-moisture levels generally favor Ralstonia solanacearum, the
exception being certain Race 3 strains that are pathogenic in potatoes and are able to grow well at lower temperatures
(Frenchet al., 1995).
FIG.4. ROTTED POTATO TUBERS OF THE PICASSO VARIETY AT FIVE DAYS AFTER INFILTRATION OF A
PATHOGEN SUSPENSION
3.4 Pathogenesis assays on potato seedlings and plants
Three potato (Solanum tuberosum) cultivars were used for pathogenicity tests: Picasso (highly sensitive), Sante (medium
resistance), and Nevskiy (high resistance). The symptoms of disease in the Picasso variety plants began to appear between
three to six days. The first symptoms of the disease were wilting leaves on the ends of branches. During disease
development, the leaves turn chlorosis and eventually become necrotic. The part of the stem close to the ground in infected
plants turns gray-brown. This is a characteristic symptom of potatoes’ brown rot (Fig.5 A and B). In the variety of Sante, the
symptoms of disease began to appear within two weeks, and the lower leaves turned browned and dry, yellowed and
chlorosis. Stems stood relatively straight for long time, and then four weeks later started to bend. The Nevskiy variety was
resistant to the pathogen-infected dose. Within six weeks there were no signs of disease. The specific symptoms were wilting
of the leaves at the end of the day with recovery at night, the edges of the leaves turning black, and curling was observed
within five to ten days, but no symptoms were observed on control plants treated with sterile water.
FIG.5: A CLOSE TO THE GROUND PART OF THE STEM OF INFECTED AN INFECTED PLANT BECAME GREY
BROWN; INFECTED PLANTS SHOW YELLOWING, WILTING, AND BROWNING OF LOWER LEAVES, FOLLOWED
BY NECROSIS.
7. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 152
3.5 Immunoblot ELISA test (Agdia).
Detection and identification of the pathogen by ELISA (Agdia product, USA) performed directly from diseased potato stems
and leaves at a concentration of 103
–104
cells/ml. Wells in which a blue color developed indicated positive results. The
bacterium was re-isolated from the infected leaves and stems, and identified as described above (Fig.6).
FIG.6. WELLS WHERE A BLUE COLOR DEVELOPED INDICATED POSITIVE RESULTS FROM DISEASED POTATO
STEMS AND LEAVES AT A CONCENTRATION OF 103
–104
CELLS/ML.
3.6 Evaluation of Streptomyces species and Trichoderma lignorium as potential antagonists against the pathogen
Ralstonia solanacerum in vitro
Primary and secondary screening by the cross-culture method have showed that the strain Streptomyces diastochromogenes
SK-6,6 inhibited the growth of Ralstonia solanacearum in 72 hours and the zone of lyses was 8.2 ± 1.25 mm. Streptomyces
bambergiensis strain SK-2-2 showed an inhibition zone — 7.9 ± 1.25 mm in the same time. Streptomyces cirratusSK-2 and
Trichoderma lignorium strains had no biological effects on Ralstonia solanacearum cultures (Table 3). When using the filter
paper and perforated agar plate methods, the antagonistic effects of Streptomyces diastochromogenes SK-6,6 and
Streptomyces bambergiensis strain SK2-2 demonstrated itself not only by suppressing the growth of the pathogen culture but
by the expression of hyper-parasitism, so that in 120 hours the growth of Ralstonia solanacearum was completely inhibited
by these antagonists ( Fig.7). Streptomyces cirratus SK-2 and Trichoderma lignoriumstrains had no biological effects on
Ralstonia solanacearum cultures in vitro bioassays. These results meant that for the following tests in soil conditions, only
two strains of Streptomyces genus were used:Streptomyces diastochromogenes SK-6,6and Streptomyces bambergiensis Sk-2-
2.
TABLE 3
MEAN DISTANCE OF INHIBITION ZONE BETWEEN EACH ANTAGONIST AND R. SOLANACEARUM FOR THE
THREE METHODS TESTED (N=3).
Treatment
Inhibition zone (mm) for the three methods tested at 72 and 120 hours
Cross streak Filter paper disk Perforated agar plate
Streptomyces bambergiensis
strain
s k -2-2
8.2 ± 1.25 Hyper-parasitism Hyper-parasitism
Streptomyces bambergiensis
strains k -1-3
7.9 ± 1.21 Hyper-parasitism Hyper-parasitism
StreptomycescirratusSK 2 No inhibition zone No effect No effect
Trichoderma lignorium No inhibition zone No effect No effect
8. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 153
FIG.5. HYPER-PARASITIC EFFECT OF STREPTOMYCES BOMBARGIENSIS (K1-3) IN 120 HOURS TO R.
SOLANACEARUM CULTURE
3.7 Effects of antagonistic strains on potato wilt in vivo
Picasso potato (high sensitivity) cultivar (Solanum tuberosum) was used for evaluation of antagonistic actinomycetes strains
Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensisstrain k1-3 in vivo tests. Soil and potato
seedlings were treated with the antagonists and their effects studied in soil conditions. Root zone and soil application of
Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensisstrain k1-3 at a dose of 108
cell/ml
significantly reduced disease incidence and increased the growth of potato plants. The disease’s progress was reduced by
60% and 56% in plants inoculated with Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensis
strain k1-3, respectively (table 4). The study revealed that sk-6 and k1-3 strains are promising strains whose effectiveness
under field conditions and their mode of action should be investigated.
TABLE 4
EFFECT OF STREPTOMYCES STRAINS CELL SUSPENSION APPLIED ON PERCENTAGE WILT INCIDENCE (PWI)
OF POTATOES IN POT EXPERIMENT
Treatments Percentage wilt incidence
Root + Soil treatment with Streptomyces diastatochromogenesstrain sk-
6 (108
cell/ml) + R. solanacearum (108
cell/ml)
34.56
(33.75)
Root + Soil treatment with Streptomyces bambergiensis strain sk-1-3
(108
cell/ml)+ R. solanacearum (108
cell/ml)
35.7
(36.2)
Inoculated control (only R. Solanacearum, (108
cell/ml)) 100.00
(99.95)
Uninoculated control (No R. solanacearum) 0.00
(0.05)
9. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 154
FIG.6. A. RECOVERY OF WILT POTATO PLANT WATERED BY BIOLOGICAL AGENT; B. IN THE CONTROL
VARIANT WITH WILT SYMPTOMS
In this study, we have used well-known, efficient methods and bioassay for systematic screening of R. solanacearum for
identification of its phenotype and biochemical profile, as well as for pathogenicity and virulence. As a result, an aggressive
race — Biovar 3 — was most isolated from the potato fields of the Tup district of the Issyk-Kul region, especially in fields
where the Picasso variety was grown. This area is characterized by a more wet and temperate climate than other areas of the
Issyk-Kul region. The low percentage of affection with this pathogenic agent was noted in the Sante variety. The pathogen
was no almost obtained from the Nevskiy variety plants and tubers. In this region, the pathogens were isolated from growing
plants with characteristic symptoms and tubers after harvest in storage, they were available for sale. In Chy region, where the
climate is hot and the humidity is relatively low (An overview of the emergence and spread of major pests and diseases,
2011), pathogenic races of R. solanacearum were obtained from the Picasso and Santa potato varieties. In this region,
essential isolates were obtained from the tubers for sale, or in storage. We have not found R. solanacearum species to be
causative agents of wilt in local potato varieties (red and white crumbly) grown in mountainous areas of the Kochkor district.
This indicates that the disease has penetrated into Kyrgyzstan from neighboring countries, together with planting material.
Our results have revealed for the first time in Kyrgyzstan the presence of the Ralstonia solanacearum bacterium as a
pathogen of bacterial wilt (quarantine for the country object) in the potato fields of the Issyk-Kul and Chy regions. Our
results have allowed us to determine which varieties are most susceptible to the disease and to which district its wide
dissemination constitutes the biggest threat. This is important for informing farmers which varieties they should buy for
planting. The areas in which commercial varieties have not yet been introduced should be remaining zones clean from this
disease.
IV. CONCLUSION
The isolated indigenous strains of Streptomyces diastatochromogenes strain sk-6 and Streptomyces bambergiensis strain k1-
3 has the potential to be used as a biocontrol agent for the management of the bacterial wilt of potatoes, as indicated by the
reduced percentage wilt incidence. The most suitable method of application of the antagonist suspension was found to be the
root + soil method. Besides biocontrol properties, the antagonist suspension applied by the root + soil method also showed
best performance in physiological and biochemical parameters indicating plant growth. However, the effective biocontrol
agent can be applied under field conditions or further commercialized only when immobilized in certain carriers. Thus,
formulations of the biocontrol agent should be prepared for easy application, storage, commercialization, and field use.
REFERENCES
[1] Smith, E.F, ―A bacterial disease of tomato, pepper, eggplant and Irish potato (Bacillus solanacearum nov.sp.)‖, USA Dep. Agric.
Div.Vegetable Physiology.Pathol.Bull.,1896, 12 pp. 1–28.
[2] Yabuuchi, E., Kosako, Y., Yano, I., Hotta, H., and Nishiuchi, Y, ―Transfer of two Burkholderia and an Alcaligenes species to
Ralstonia Gen. Nov.: proposal of Ralstonia pickettii and Ralstonia eutropha‖, Microbiol. Immunol.,1995, 39, pp.897–904
10. International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016]
Page | 155
[3] Allen C., Prior, P and Hayward, A.C,‖Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex‖, APS Press, St Paul,
MN, USA,2005, pp. 528.
[4] Agrios, G.N. Plant Pathology, 4th Edition. Academic Press, San Diego, CA,1997.
[5] Stevenson, W.R., R. Loria, G.D. Franc, and D.P. Weingartner, Eds. Compendium of Potato Diseases, 2nd Ed. APS Press, St. Paul,
MN. 2001.
[6] Staskawicz, B.J., Mudgett, M.B., Dangl, J.L., and Galan, J.E,―Common and contrasting themes of plant and animal diseases. Science,
2001, 292, pp.2285–2289.
[7] Granada, G. A. and Sequeira, L, ―Survival of Pseudomonas solanacearum in soil, rhizosphere and plant roots‖, Can. J. Microbiol.,
1983, 29, pp.433–440.
[8] Genin, S and Denny, T.P, ―Pathogenomics of the Ralstonia solanacearum species complex‖, Ann. Rev. Phytopatholy, 2012, 50, pp.
67–89.
[9] French, E.B., L. Gutarra, P. Aley, and J. Elphinstone, ―Culture media for Ralstonia solanacearum isolation, identification, and
maintenance‖, Fitopatologia,1995, 30 (3), pp. 126–130.
[10] Daughtrey, M, ―Southern bacterial wilt, caused by Ralstonia solanacearum‖, Society of American Florists’ 19th Annual Conference
on Insect and Disease Management on Ornamentals, 2003
[11] Jaunet, T.X. and J.F. Wang, ―Variation in genotype and aggressiveness of Ralstonia solanacearum race 1 isolated from tomato in
Taiwan‖, Phytopathology, 1999, 89, pp. 320–327.
[12] Kim, S.H., T.N. Olson, N.W. Schaad and G.W. Moorman, ―Ralstonia solanacearum race 3, biovar 2, the causal agent of brown rot of
potato, identified in geraniums in Pennsylvania, Delaware, and Connecticut. Plant Dis, 2003, 87,pp.450.
[13] Salanoubat M., Genin, S., Artiguenave F., Gouzy. J., S, ― Mangenot. Genome sequence of the plant pathogen Ralstonia
solanacearum‖, Nature, 2002, Vol. 415, pp.497–502.
[14] Grimault, V., J. Schmit and P. Prior, ―Some characteristics involved in bacterial wilt (Pseudomonas solanacearum) resistance in
tomato‖, In: Hartman, G.L. and A.C. Hayward (eds.), Bacterial Wilt, 1993 pp.112. ACIAR Proceedings, No. 45: Australian Centre for
International Agricultural Research, Camera.
[15] Shekhawat, G.S., S.K. Chakrabarti, V. Kishore, V. Sunaina, and A.V. Gadewar, ―Possibilities of biological management of potato
bacterial wilt with strains of Bacillus sp., Bacillus. subtilis, Pseudomonas fluorescens and Actinomycetes‖, In: Hartman, G.L. and
A.C. Hayward (eds.), Bacterial Wilt, 1993. pp. 327,ACIAR Proceedings, No. 45: Australian Centre for International Agricultural
Research, Camera.
[16] Higa, T, ―Effective microorganisms — A holistic technology for humankind‖, In: Senanayake, Y.D.A. and U.R. Sangakkara (eds.),
Proceeding of Fix International Conference on Kyusei Nature Farming,1999, pp: 19–28.
[17] Lwin M.M. and Ranamukhaarachch, ―Development of Biological Control of Ralstonia solanacearum Through Antagonistic Microbial
Populations‖. International Journal of agriculture and biology,2006, Vol. 8, No. 5, pp.657–660.
[18] Hussain, T., Ahmad R., Jillana G., Yaseen M., and Akhtar S.N. Applied EM Technology, 1993, pp.1–6, Nature Farm Research
Center, University Agriculture, Faisalabad, Pakistan.
[19] GuoJ.H., Qi H.Y, GuoY.H, Ge H.L., Gong L.Y., Zhang L.X., and Sun P.H, ― Biocontrol of tomato wilt by plant growth-promoting
rhizobacteria‖,Biological Control, 2004, Volume 29, Issue 1, pp. 66–72.
[20] Lemessa F., Zeller W., ―Screening rhizobacteria for biological control of Ralstonia solanacearum in Ethiopia‖, Biological Control,
2007, Volume 42, Issue 3, pp. 336–344.
[21] Toyota K., Kimura M. ―Growth of the bacterial wilt pathogen Pseudomonas solanacearum introduced into soil colonized by
individual soil bacteria‖. Soil Biology and Biochemistry, 1996, 28, pp. 1489–149.
[22] Nguyen M.T. and Ranamukhaarachchi S.L, ― Soil-borne antagonists for biological control of bacterial wilt disease caused by
Ralstonia solanacearum in Tomato and Pepper‖, Journal of Plant Pathology,2010, 92 (2), pp.395–406
[23] An overview of the emergence and spread of major pests and diseases of crops in the Kyrgyz Republic in 2010 and the forecast of
their appearance in 2011. Bishkek.
[24] http://www.kartofel.org/bolezn/bacteria/bacwilt.htm
[25] French, E.B., Gutarra L., Aley P., and Elphinstone J., ―Culture media for Ralstonia solanacearum isolation, identification, and
maintenance‖, Fitopatologia, 1995, 30 (3), pp.126–130.
[26] Staskawicz, B.J., Mudgett, M.B., Dangl, J.L. and Galan, J.E. ―Common and contrasting themes of plant and animal diseases‖,
Science, 2001, 292, pp. 2285–2289.
[27] Kim, S.H., Olson T.N., Schaad N.W. and G.W. Moorman, ―Ralstonia solanacearum race 3, biovar 2, the causal agent of brown rot of
potato, identified in geraniums in Pennsylvania, Delaware, and Connecticut‖, Plant Dis.,2003, 87, pp. 450.
[28] Daughtrey, M,‖Southern bacterial wilt, caused by Ralstonia solanacearum‖, Society of American Florists’ 19th Annual Conference on
Insect and Disease Management on Ornamentals, 2003.
[29] Fegan, M. and Prior, P.‖How complex is the ―Ralstonia solanacearum species complex‖? in: Bacterial Wilt Disease and the Ralstonia
solanacearum Species Complex. C. Allen, P. Prior, and A. C. Hayward, eds. American Phytopathological Society, St. Paul,
MN.,2005, pp. 449–461.
[30] Crawley, Michael J., Methods in Ecology: GLIM for Ecologists. Oxford Blackwell Scientific publications, London. 1995.