Tetrazolium Chloride Medium Research Articles

Inhibition of Ralstonia solanacearum by Warburgia ugandensis stem bark and leaf crude extracts obtained using organic solvents

Ralstonia solanacearum is a soil-borne bacterial pathogen that poses significant threat to the Solanaceae family and other crops. It causes widespread bacterial wilt, a devastating disease that affects the plant's water transport system, leading to wilting and death. Numerous chemical agents and treatment methods have been employed in attempts to control R. solanacearum, but are ineffective. The study aimed to determine the in vitro efficacy of W. ugandensis stem bark and leaf crude extracts against R. solanacearum. W. ugandensis stem bark and leaf crude extracts were obtained using organic solvents viz. methanol, ethanol, dichloromethane and hexane. In vitro, antagonistic activities against R. solanacearum of all organic crude extracts of W. ugandensis were determined by standard agar well diffusion assay on Kelman’s 2, 3, 5- triphenyl tetrazolium chloride medium in triplicates. Two-way analysis of variance (ANOVA) was used in the statistical analysis of the mean diameter inhibition zones. All the organic solvents crude extracts of W. ugandensis were inhibitive against R. solanacearum. However, the stem bark crude extracts exhibited significantly higher efficacy against R. solanacearum compared to the leaf crude extracts. The crude extracts were subjected to a serial dilution to determine the minimum inhibitory concentration (MIC). W. ugandensis stem bark dichloromethane crude extracts had the lowest MIC of 1 mg/ml. W. ugandensis stem bark dichloromethane crude extracts were most effective against R. solanacearum. Further research is important to determine the bioactive compounds against R. solanacearum in W. ugandensis stem bark dichloromethane crude extracts.

First Report of Ralstonia pseudosolanacearum Causing Wilt Disease in Tomato (Solanum lycopersicum L.) Plants from Mexico.

Mexico produces more than four million tons of tomato fruits and ranks tenth worldwide. In February 2022, tomato plants in a greenhouse in Culiacan, Sinaloa State, were affected by wilt diseases with an incidence of 20% and irreversible wilt and death of the infected plants (severity up 70%). When cut stems from affected plants, a reddish to brown discoloration of the vascular system was observed and these were disinfected with 1% NaClO for 5 min and then placed in a humid chamber. Characteristic milky-white exudate was obtained. From that exudate, irregular, mucoid, and white colonies with pink centres were obtained on casamino peptone glucose (CPG) plates supplemented with 1% 2,3,5-triphenyl 15 tetrazolium chloride (TZC); these characteristics are typical of the Ralstonia solanacearum species complex (RSSC) (Garcia et al., 2019). Identification of the pathogen was done by PCR using specific primer pairs reported by Paudel et al. (2022), RssC-wF3 (5'-TATATATCCTCGACTTTTCCATGAAGCTGTG-3') - RssCwR3 (5'-CTATATATATACCCCACTTGTTGAGGAACTG-3') and Rpseu-wF5 (5'-TTTTATTTTTTTGGTGTCCGGGCCAAGATAG-3') - Rpseu-wR5 (5'- TTATATTACTCGAACGTGCTGCAAAACCACT-3'), which amplified fragments of 162 and 251 bp for RSSC and Ralstonia pseudosolanacearum, respectively. Additionally, 759 (5'-GTCGCCGTCAACTCACTTTCC-3') - 760 (5'-GTCGCCGTCAGCAATGCGGAATCG-3') (Opina, et al., 1997) and Nmult21:1F (5'-CGTTGATGAGGCGCGCAATTT-3') - Nmult22:RR (5'- TCGCTTGACCCTATAACGAGTA-3') (Fegan and Prior, 2005) were used to generate 282 and 144 bp amplicons for RSSC and phylotype I, respectively. Subsequen to making the specific detection, the representative strain ClnMx was used to generate a sequence for the endoglucanase (egl) gene for separation into sequevars by using the primers Endo-F (5'- ATGCATGCCGCTGGTCGCCGC-3') and Endo-R (5'-GCGTTGCCCGGCACGAACACC-3'), which amplified a fragment of 750 bp (Fegan et al., 1998). The egl sequence (GenBank Access ON542479) showed 100% identity with the well-defined R. pseudosolanacearum sequevar 14, which was isolated from tomato plants from Senegal (UW763, I-14 GenBank Access CP051174) (Steidl et al., 2021), as well as, the strain MAFF 301070 (GenBank Access AB508612) from Japanese tomato. For pathogenicity tests, four 1-month-old tomato plants were infected using an insulin syringe that contained a pure bacterial suspension with approximately 2x108 CFU/mL. For each plant, 20 µL was infiltrated into the axil of the third upper leaf, and for untreated controls, tomato plants were infiltrated with sterile water. All plants were kept at 28°C under greenhouse conditions. Symptoms resembling those observed in the field were observed in inoculated plants six days after inoculation, and the plant pathogen was recovered on TZC medium. To confirm the bacteria identification a PCR using the specific primer pairs mentioned early was carried out. In contrast, water-treated control plants remained healthy. Koch's postulates were carried out twice with similar results. Ralstonia solanacearum species complex (RSSC) causes severe economic losses in many countries of the world because of their capability to infect a wide range of host plants, including potato, tomato, eggplant, tobacco, and, banana, among others. Ralstonia pseudosolanacearum has been reported to cause tomato wilt disease mainly on the Afro-Eurasian continent in areas such as Senegal, Cambodia, and Japan (Klass et al., 2019). To our knowledge, this is the first report of R. pseudosolanacearum causing bacterial wilt diseases in tomato plants from Mexico and because, the control of this bacteria is a challenge by the long survival time in soil, water, and infected plant tissues, the identification of this important pathogen could provide relevant information for developing management strategies.

Bacterial Wilt of Sacha Inchi (Plukenetia volubilis) Caused by Ralstonia pseudosolanacearum Phylotype I in Southern China

Sacha inchi (Plukenetia volubilis L.) is a little-known yet important perennial plant native to the Andes Mountains of Peru. It is a plant producing large, edible seeds rich in omega-3 fatty acids and proteins useful for promoting human health. The economic importance of sacha inchi has increased in recent years because its seeds produce significantly higher oil yields than those of other plants (Wong 2018). Sacha inchi was first introduced to grow in Xishuangbanna, Yunnan Province, southwest China in 2006, and then to Lingshui County, Hainan Province, southern China in 2014. However, just 2 years later, in April 2016, unusual wilting was observed on many sacha inchi plants in the fields of Lingshui County. The disease outbreak proved to be rapid and extensive, covering approximately 11 ha of sacha inchi growth and causing an 80% production loss in that crop’s area in just a few months. By 2017, the farmers had almost given up on continuing sacha inchi production owing to the severity and spread of this disease. In the fields, the disease caused the plant’s stem and leaves to wilt. On seedlings, their leaves wilted and drooped while still being photosynthetically active (i.e., green), followed by total plant collapse within a few days. Mature plants initially had symptoms of irregular, black necrotic lesions at their leaf base margins. But as the disease symptoms progressed, the necrotic leaves spread upward, and eventually the affected plants turned chlorotic and shed leaves and developed black stripes on their stems. Vascular necrosis and bacterial ooze were observed when longitudinal sections were obtained from the basal portion of infected stems. For identification, a total of 10 plants with the typical wilting symptoms were collected from the sacha inchi fields of Lingshui County. Six isolates were obtained from the roots and plantlets of six separate plants. All the isolates were gram negative, oblong to rod shaped, and 0.4 to 0.9 × 0.7 to 2.0 μm in size (n = 25) when viewed under electron microscopy. When cultured on Kelman’s tetrazolium chloride medium, the colonies appeared round to oval, fluidal, and entirely white with a pale red center after incubation at 30°C for 48 h. Three Ralstonia solanacearum-specific primer pairs, for the flagella subunit (Rsol_fliC-F/ Rsol_fliC-R) (Schonfeld et al. 2003), the polygalacturonase gene (pehA #3/ pehA #6) (Gillings et al. 1993), and 759/760 (Ito et al. 1998) yielded the expected amplicons. Two representative strains (ACCC60145 plu-3 and ACCC60146 plu-6) were identified as R. pseudosolanacearum phylotype I, sequevar 34, according to the phylotype-specific multiplex polymerase chain reaction (PCR) assay (Fegan and Prior 2005) and phylogenetic analysis of the partial egl gene sequences (GenBank accession nos. KY352419 and KY352421) (Safni et al. 2014). The two strains shared 99.9 and 100% sequence identity with R. pseudosolanacearum GMI1000 (AL646052), which had been identified using 16S ribosomal RNA (GenBank accession nos. KY346975 and KY346977) sequencing and sequence comparisons. For the pathogenicity test, stems of 30 1-month-old sacha inchi (cv. Pinto Recodo) seedlings were injected with 40-μl suspensions (10⁸ CFU/ml) of the representative isolates ACCC60145 plu-3 and ACCC60146 plu-6 (10 seedlings per isolate inoculation treatment). The injection point on the seedling stem was at 2 to 3 cm above the soil surface. Symptoms of wilt were observed 5 days after these inoculations of sacha inchi. To serve as a negative control, 20 healthy sacha inchi seedlings were likewise injected but with sterile water. No disease symptoms were observed on these control plants. Reisolations were done twice, as outlined above, on the symptomatic seedlings and control plants at 3 to 4 cm above the original injection points. To complete Koch’s postulates, the bacteria were then reisolated from the inoculated sacha inchi plants and reidentified by PCR. Following the same inoculation procedure, the bacterium also caused wilting in cherry tomato (Lycopersicon esculentum Mill.; 20 of 20 individuals), eggplant (Solanum melongena L. var. esculentum Nees.; 17 of 20), and pepper (Capsicum annuum var. conoide Mill. Irish; 18 of 20). To the best of our knowledge, this is the first report of R. pseudosolanacearum phylotype I sequevar 34 causing bacterial wilt of sacha inchi in China and around the world. These disease strain findings can be useful for developing effective strategies for control of the disease in the important oil and seed plant sacha inchi.

Uji In Vitro Asap Cair terhadap Ralstonia syzygii subsp. celebesensis Penyebab Penyakit Darah pada Pisang

In Vitro Test of Liquid Smoke against Ralstonia syzygii subsp. Celebesensis, the Cause of Blood Disease in Bananas Blood disease, caused by Ralstonia syzygii subsp. celebesensis, is an important disease of banana plants in Indonesia. Several control methods have been done, but the result were not effective. Liquid smoke from wood waste is reported to be antimicrobial, but its use for controlling R. syzygii subsp. celebesensis has never been reported. Research was conducted to examine the ability of liquid smoke produced from coconut shell (CS-LS), pinecone (P-LS), and oil palm branch (OPB-LS) in inhibiting the growth of R. syzygii subsp. celebensensis in vitro and its effect on bacterial cell morphology. Efficacy test of liquid smoke was carried out by agar diffusion method and measurement of bacterial density by spectrophotometry with ʎ 600 nm. Observation of bacterial cell morphology was carried out by electron microscopy. The CS-LS, P-LS, and OPB-LS starting from 0.5% concentration showed inhibitory activity against R. syzygii subsp. celebesensis both on triphenyl tetrazolium chloride medium and luria bertani broth. Liquid smoke also caused damage to cell walls and cell membranes. Therefore, liquid smoke has the potential to be used as component in control method for R. syzygii subsp. celebesensis.

Isolation, identification and molecular characterization of Ralstonia solanacerum isolates collected from Southern Karnataka

Bacterial wilt caused by Ralstonia solanacearum, is the major threat to tomato cultivation in all tomato growing areas of Karnataka. R. solanacearum was isolated from the infected host plants collected from different locations of southern Karnataka. The identity of the isolates was established using morphological, biochemical, and molecular analysis using species specific PCR primers. The race and biovar specificity of pathogen was determined through pathogenicity test on different host plants and the ability of isolates to use carbohydrates, respectively. Phylotype classification was done by phylotype specific multiplex PCR using phylotype specific primers. All the bacterial isolates showed the characteristic creamy white fluidal growth with pink centre on the Tetrazolium chloride medium. Further, the isolates amplified at 280 bp, which confirmed the identity of pathogen as Ralstonia solanacearum. Our results showed that all isolates belonged to Race 1 of the pathogen. Among different isolates obtained, four isolates each were identified to be Biovar III and Biovar IIIA, repectively, while two isolates were identified as Biovar IIIB. All the ten isolates were affiliated to Phylotype I of Ralstonia solanaceraum species complex. These findings may help in devising the management practices for bacterial wilt of tomato in southern Karnataka.

BIO-CONTROL OF POTATO BACTERIAL WILT CAUSED BY Ralstonia solanacearum ISOLATED FROM DIFFERENT SOURCES

Seventeen pathogenic isolates of brown rot pathogen were collected from field soil, potato tubers, weeds and irrigation water at major potato districts in Egypt. Isolates recovered produced pink, or light red, colour with whitish margin on tetrazolium chloride medium (TZC) indicated possible virulent Ralstonia solanacearum pathogenic isolates. Pathogenicity test revealed the ability of the isolates to cause wilt symptoms in potted potato plant and three leaved tomato seedlings. Morphological, physiological and biochemical tests were used in identification. The biovar determination was made using for oxidization of disaccharides (maltose, lactose, and cellobiose) and confirmed the identity to R. solanacearum, race 3, biovar II equivalent to phylotype II, sequevar 1. A polymerase chain reaction (PCR) confirmed the identity of highest virulent isolates from tuber (T6) and from water (W11) to R. solanacearum. The application of biological agents Pseudomonas fluorescens, Bacillus subtilis, Pseudomonas aeruginosa andTrichoderma spp. decreased R. solanacearuminfected plants and caused greater decrease in severity.

English

Bacterial wilt disease has caused a serious threat to the banana industry in Malaysia. The disease is always associated with Moko disease caused by Ralstonia solanacearum. The bacterium forms irregular round, creamy colonies with red centres when cultured on Kelman’s tetrazolium chloride (TZC) medium. Moko disease has been interchangeably identified as blood disease as both of them cause similar wilting symptoms on banana. Therefore, the present study was carried out to detect the occurrence of bacterial wilt in Selangor through comprehensive procedures on morphology, molecular, pathogenicity and specificity tests. Isolation of bacteria was conducted on infected plant samples (fruit stalk, fruit pulps, stem and root) collected in the field by plating on TZC medium. Bacteria colonies that are Gram-negative, oxidase-positive, and obligate aerobe were chosen for further identification through polymerase chain reaction (PCR) using 759F and 760R primers. Molecular identification showed the bacteria that colonized the infected plants is confirmed as BDB, which is very closely related to R. solanacearum. After Koch’s Postulate and pathogenicity test on tomato, wilting symptoms were found on all banana plantlets after two weeks. The bacterium did not cause any wilting symptom on tomato plantlets. The present study revealed the distribution of blood disease in Selangor, after the first report in Perak. It also suggested that both morphology and DNA-based identification are necessary for the detection of the pathogen that causes bacterial wilt in banana. © 2016 Friends Science Publishers

First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Chard in Taiwan.

Chard (Beta vulgaris var. cicla L.) is a biennial herbaceous plant in the Chenopodiaceae family. It is rich in vitamins and minerals and is one of the most popular traditional vegetables in Taiwan. Chard accessions VI048530 and VI050121 growing in fields at Shanhua, Tainan, showed wilting symptoms in March and April 2013. The initial symptoms of wilt were observed on young green leaves. These symptoms progressed over time to chlorosis, interveinal necrosis, and finally blight. Finally, the plants collapsed and died. Vascular and pith tissues were discolored, especially at the stem base. A whitish mass oozed from the cut end of diseased stems. A total of eight bacterial strains were isolated from stems and roots of wilted chard plants. On tetrazolium chloride (TZC) medium (4), colonies were round to oval, fluidal, and white with a pink or red center after incubation at 30°C for 48 h. A typical hypersensitive reaction was induced within 24 h when the strains were infiltrated into tobacco leaves. Koch's postulates on chard plants were confirmed using the eight strains within a greenhouse, under natural light, with temperature and humidity ranges from 25 to 34°C and 56 to 98%, respectively. Fifteen chard (VI048530) plants at the four- to six-leaf stage were inoculated by soil drenching with 30 ml of a ~108 CFU/ml bacterial suspension. Sterile water was used as negative control. After 4 to 6 days, the first symptoms of wilt were observed on the young chard leaves. The progression of symptom development was identical to that observed in the field. The colony morphology on TZC medium of isolates from the inoculated plants was identical to that previously described from field samples. Pathogenicity of the strains was also tested on tomato (VI005790), eggplant (VI046095), and pepper (PBC1367) plants using the previously described inoculation procedure. The mean disease incidences on tomato, eggplant, and pepper plants were 100% (120/120), 100% (120/120), and 79.2% (95/120) respectively. Latent infection was found in asymptomatic pepper plants (16/120) by a printing method. Polymerase chain reaction (PCR) amplification of total DNA from each strain using the Ralstonia solanacearum-specific primer pair AU759f and AU760r (5) produced the expected 282-bp amplicon. All the isolated strains were identified as biovar 3 based on their capacity to utilize three hexose alcohols (mannitol, sorbitol, and dulcitol) and three disaccharides (lactose, maltose, and cellobiose) (2) to produce acid. Based on the phylotype-specific multiplex PCR assay and the partial egl gene sequence (GenBank accession numbers KM100442 to KM100449) (1), all chard isolates were identified as R. solanacearum phylotype I, sequevar 34. Bacterial wilt symptoms have also been observed on beet (Beta vulgaris L.), a close relative of chard, but beet has not been confirmed as a host plant (3). To our knowledge, this is the first report of chard as a host of R. solanacearum worldwide.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum Biovar 2 Race 1 on Tomato in Egypt.

This study aims to isolate and identify the causal pathogen of tomato bacterial wilt in Egypt. In 2008, tomato plants showing typical symptoms of bacterial wilt disease with no foliar yellowing were observed in Minia, Assiut and Sohag governorates, Egypt. When cut stems of symptomatic plants were submerged in water, whitish ooze was evident and longitudinal sections showed a brown discoloration in the vascular tissues. Bacteria were isolated on triphenyl tetrazolium chloride medium and fifteen isolates shown typical morphological and cultural characteristics were confirmed as Ralstonia solanacearum biovar 2 race 1. Pathogenicity tests showed that all isolates proved to be pathogenic to tomato plants, varied from 52 to 97% wilting. This is the first report of R. solanacearum biovar 2 race 1 causing bacterial wilt in tomato crop in Egypt.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Roselle in Taiwan.

Roselle (Hibiscus sabdariffa L.) is a herbaceous plant belonging to the Malvaceae family. Its calyxes are rich in vitamin C and anthocyanins and are used to make roselle drink and hibiscus tea. Roselles are grown in counties of Taitung, Pingtung, and Chiayi in Taiwan. In addition to a few local cultivars, the major cultivar currently grown in Taiwan is Roselle cv. Victor. In April of 2012, a wilt disease appeared on seedlings of a cultivar, Chiada 1, at the Chungpu Township of Chiayi County. Mature plants were free from this disease. Leaves appeared weak and drooping when they were still green, followed by collapse of the whole plant a few days later. Browning of vascular and pith tissues was evident, especially at the base of the stem. A whitish mass of bacteria oozed from the cut end of diseased stems, suggesting that bacteria might be the cause of this disease. A total of 15 bacterial strains were collected. Colonies on tetrazolium chloride medium (3) were round to oval and fluidal, each with a pink or red center after incubation at 30°C for 48 h. When tobacco leaves were infiltrated with these strains, a hypersensitive reaction (HR) typical of phytopathogenic bacteria was induced. All strains produced the expected amplicon (282 bp) after PCR with the Ralstonia solanacearum-specific primer pair, AU759f and AU760r (4). Three hexose alcohols (mannitol, sorbitol, and dulcitol), rather than three disaccharides (lactose, maltose, and cellobiose), were utilized, which suggests R. solanacearum biovar 4 (2). R. solanacearum phylotype I was determined by phylotype-specific multiplex PCR (1). Pathogenicity of the strains was tested on roselle, tomato, pepper, and eggplant. Young plants of the various species were inoculated at the four- to six-leaf stage by soil drenching with 30 ml of bacterial suspension (about 108 CFU/ml). Control plants were inoculated with sterile water. Each treatment comprised eight plants with a single plant in each pot. Plants were incubated in a greenhouse at 25 to 31°C and 56 to 93% humidity. Wilting was observed 4 to 6 days after inoculation, while the control did not wilt. To find the correlation between plant growth stage and resistance to the pathogen, 2-, 3-, 4-, and 5-week-old roselle plants cv. Chiada 1 were transplanted into artificially infested soil. Eight plants in each treatment were planted with a single plant in each pot. The disease incidences for plants of different ages were 75%, 62.5%, 50%, and 12.5%, respectively. This study showed that resistance increases with plant age. Hence, if older seedlings are transplanted, the risk of bacterial wilt of roselle can be reduced. To our knowledge, this is the first report of R. solanacearum on roselle in Taiwan.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Mesona chinensis in China.

Jellywort (Mesona chinensis Benth) is an herbaceous plant in the Lamiaceae (mint) family. The plant is referred to as 'Xiancao' (weed from angels) in Chinese and primarily used to make grass jelly, a popular refreshing drink. Currently, Xiancao cultivation is a fast-growing industry with a high profit margin in southern China. An estimated 7,000 ha is grown with a value of more than $50 million USD. In June, 2009, a wilting disease of Xiancao was observed in Guangdong and the neighboring Guangxi and Fujian provinces with incidence up to 50% in the severest case. Affected plants initially show withering symptoms on apical leaves during the daytime with recovery at night. As the disease develops, withering leaves spread downward, eventually encompassing the whole plant. Leaves lose vigor but remain green. After 3 to 4 days, wilting becomes irreversible. Roots and basal stem tissues blacken and rot, leading to plant death. Longitudinal sectioning of the basal stem shows browning of vascular tissues with whitish ooze visible when compressed. To investigate the disease etiology, 12 Xiancao plants from three cultivars showing typical wilting symptoms were collected from a production field in Zengcheng City of Guangdong Province in June 2010. A total of 27 bacterial isolates showing large, elevated, and fluidal colonies with a pale red center were isolated from vascular tissue on tripheny tetrazolium chloride medium (3) after incubation at 30° for 2 days. Fifteen 45-day-old Xiancao plants (cv. Zhengcheng 1) were inoculated by injection of 20 μl of bacterial suspension (1 × 108 CFU/ml) into the middle stem. Sterile water was used as a negative control. After 4 to 6 days of incubation in a greenhouse (28 to 30°), all (15 of 15) inoculated plants developed wilting symptoms as described above. The same bacterium was reisolated from inoculated plants. The five negative control plants did not show any wilting symptoms. With the same artificial inoculation procedure, this bacterium also caused similar wilting disease in tobacco, potato, tomato, pepper, and eggplant. An inoculation test with a tomato strain of Ralstonia solanacearum resulted in similar symptoms. On the basis of symptomatology and bacterial culture characteristics, R. solanacearum (formerly Pseudomonas solanacearum) was suspected as the causal agent. For confirmation, the universal bacterial 16S rDNA primer set E8F/E1115R (1) was used to amplify DNA from pure culture. A 1,027-bp DNA sequence was obtained and deposited in GenBank with Accession No. HQ159392. BLAST search against the current version of GenBank database showed 100% similarity with the 16S rDNA sequences of 26 R. solanacearum strains. Furthermore, primer set 759/760 (4) amplified a specific 280-bp fragment. Along with the result from multiplex PCR (2), the bacterium was identified as R. solanacearum Phylotype I. To our knowledge, this is the first report of a disease caused by R. solanacearum on M. chinensis.

Bacterial Wilt of Solanaceae Caused by Ralstonia solanacearum Race 1 Biovar 3 in Mali.

Ralstonia solanacearum (Smith) Yabuuchi et al. causes bacterial wilt worldwide on a wide range of plant species. In Mali, the disease is commonly found on potato (Solanum tuberosum L.), tomato (Lycopersicon esculentum var. esculentum L.), pepper (Capsicum annuum L.), eggplant (Solanum melongena L.), tobacco (Nicotiana tabacum L.), and peanut (Arachis hypogaea L.). Determination of race and biovar is critical for development of potato seed certification programs for management of the disease. Isolates (25) of R. solanacearum were obtained from wilting potato, pepper, eggplant, tobacco, and tomato plants collected from fields near Baguineda, Sonityeni, Sotuba, Sikasso, and Kolikoro. Isolations were made from bacterial streaming by dilution plating on triphenyl tetrazolium chloride medium (TZC) (2). Characteristic colonies were selected and identified by ELISA or Immunostrips (Pathoscreen Rs, Agdia Inc., Elkhart, IN). These isolates were used in host range studies and hypersensitivity (HR) tests on tobacco (cv. xanthi) (3) and tested for their ability to produce acids on Ayers basal media amended with disaccharide and hexose alcohol carbon sources (1). These isolates caused characteristic wilt 40 days postinoculation on greenhouse-grown tobacco (cv. Xanthi), peanut (cv. 4610), and tomato (cv. Roma VF) plants when stems of five plants of each host were syringe inoculated with 0.1 ml of a 1 × 109 CFU/ml of bacteria. Plants inoculated with sterile distilled water remained symptomless and R. solanacearum was reisolated from infected plants on TZC and identified with Immunostrips. All HR tests were negative. Infection of peanut, tobacco, and tomato and the results of the HR tests indicated that all isolates were Race 1 and no significant variation was noted between isolates. Acid was produced from the hexose alcohols: mannitol, sorbitol, and dulcitol; and the disaccharides: cellobiose, lactose, and maltose. This indicated that all isolates were biovar 3, the same as a known Race 1 strain from tobacco (MSU Plant Pathology teaching collection) (1). To assess relative distribution of R. solanacearum, 20 soil samples collected from potato fields in the vicinity of Baguineda, Kati, Koulikoro, and Sikasso were placed in pots (30 × 25 cm) under shade cloth at the IER Station in Sotuba and planted with 30-day-old tobacco plants. After 90 days, infected plants (35 to 100% infection) were found in all soils. Infected plants exhibited classical wilt symptoms and tested positive for R. solanacearum infections as confirmed by Immunostrip tests. Six of nine surface water samples taken near potato fields in Baguineda, Sikasso, Mopti, and Koulikoro tested positive for the presence of R. solanacearum by an Agdia Inc. enrichment kit and ELISA. A weed, Commelina forskalaei (Vahl), collected by Farako creek near Sikasso tested positive in the Immunostrip test even though no symptoms were obvious. No attempt was made to characterize the race, biovar, or phylotype of the soil, water, and weed isolates. To our knowledge, this is the first time that the race and biovar of R. solanacearum from Mali has been reported and the wide distribution of this pathogen in Malian soils and surface water has been demonstrated. It is significant that we did not detect Race 3 biovar 2, which is subject to quarantine and biosecurity regulations.

Study on intraspecific diversity of Ralstonia solanacearum strains in West Malaysia using whole cell fatty acid analysis

Surveys were conducted between the years of 2005 and 2006 at several locations in the northern, central and southern parts of West Malaysia to study the polymorphism of Ralstonia solanacearum strains. These sites included vegetables and farms with known hosts of the pathogen, such as banana, tomato, eggplant, chili and tobacco. Samples were collected from the suspected wilted plants and weeds, including soil and water samples, in selected areas. The bacterium was isolated in all samples using semi-selective tetrazolium chloride medium (TZC). The bacteria strains were detected by using the BIOLOG identification system and were confirmed by nested-PCR. Fatty Acid Methyl Esters (FAME) profiling was performed to determine polymorphism among 58 bacterial isolates. The results showed that the fatty acid composition varied for all R. solanacearum isolates. Grouping of R. solanacearum isolates by fatty acid composition suggested that the existence of distinct groups that were significantly related to host of bacteria but low correlation between fatty acid profiles and biovar or sampling site was detected. A unique FAME profile was found among the strains that have been isolated from banana.

Discovery of an atypical strain ofBurkholderia solanacearum affecting potatoes in India

An atypical strain ofBurkholderia solanacearum was isolated from potato. Characters examined were colony shape, size, colouration, pathogenicity to susceptible hosts, vascular discolouration and biovar features. In contrast to normal white-coloured colonies, this atypical strain produced cream coloured, fluidal colonies on tetrazolium chloride medium. It did not cause disease symptoms in eggplant, datura and pepper but was slightly to highly pathogenic on tomato and potato respectively but caused no discolouration of vascular tissues. This atypical strain from potato also differed from established biovars in its ability to produce acid only from mannitol and maltose, a combination which has not been previously reported. This is believed to be the first report of the presence of an atypical strain ofB. solanacearum from India.

Protection of Tomato Seedlings by Pre-treatment with Pseudomonas glumae from Infection with Pseudomonas solanacearum and Its Mechanisms.

Pseudomonas glumae strains showed different antibiotic activities against P. solanacearum depending upon kind of media used. When triphenyl tetrazolium chloride medium (TTC medium) was used, all strains of P. glumae formed growth inhibition zones around their colonies on the lawn of P. solanacearum used as indicator. Pre-treatment of the roots of tomato seedlings with the suspension of antibiotic productive strains of P. glumae suppressed the severity of wilt incited by challenge inoculation with P. solanacearum. The disease suppression was positively correlated with length of dipping time and concentration of the bacterial suspension used for pre-treatment. Dipping of the roots of tomato seedlings into the bacterial suspension of ca. 1010cfu/ml for 24hr showed highest suppressiveness. The pre-treatment of tomato roots with non-antibiotic-producing strain or heat-killed cells of P. glumae also showed protection, suggesting that some mechanisms other than antibiotic productivity were involved in the suppression of the disease.

Response of Wheat and Barley of Azospirillum brasilense Inoculation

Summary The associative symbiosis of five Azospirillum brasilense strains isolated from different ecosystems with twelve cultivars of wheat and barley commonly cultivated in Egypt was investigated. Inoculation significantly increased N 2 -ase activity in all cultivars particularly in the last two periods except with barley cultivars Giza 257/36, C.C. 89 and Younis. The highest increases in dry weight were recorded when the plants inoculated with the bacterial isolates V and Sp Br 14 where the increases were 1.1–28.7 and 2.1–26.2% over the corresponding controls, respectively. The maximum amounts of indole acetic acid in the bacterial cultures (4.8–8.5 and 4.5–8.8 mg/l) were detected during the 10- and 15-day periods, respectively. After overnight incubation of surface sterilized roots in malate-phosphate buffer — 2,3,5-triphenyl tetrazolium chloride medium, all the inoculated plants developed a more intensive red colour than the uninoculated correspondings.

Methods of Preservation of Corynebacterium insidiosum Isolates in Relation to Virulence and Colony Appearance on a Tetrazolium Chloride Medium

Methods of Preservation of Corynebacterium insidiosum Isolates in Relation to Virulence and Colony Appearance on a Tetrazolium Chloride Medium

Use of an improved cetrimide agar medium and other culture methods for Pseudomonas aeruginosa.

In a comparison of two selective media for Ps. aeruginosa containing 0.03% cetrimide, stronger fluorescence was obtained from growth on the medium prepared with King's medium B as the base (CTA 2); although Ps. aeruginosa was not isolated more frequently from burns on this medium than from cetrimide agar made with a Lemco base (CTA 1), results were easier to assess and there were fewer cases of doubtful fluorescence on CTA 2 than on CTA 1. Pyocyanin production on CTA 1 was better than on CTA 2, but for demonstration of pyocyanin production Wahba and Darrell's medium was preferable and showed the pigment in 455/497 (92%) of fluorescent strains of Ps. aeruginosa from burns.A series of tests on 99 strains of Ps. aeruginosa or presumptive Ps. aeruginosa, on 30 strains of other species of Pseudomonas, and on 68 strains of other species of Gram-negative rods showed the value of growth on cetrimide agar and on triphenyl tetrazolium chloride medium, of slime production in gluconate broth, of gas production from nitrate, and of growth at 42 degrees C. as diagnostic aids in the recognition of Ps. aeruginosa.