First report of Xanthomonas campestris pv. campestris as the causal agent of necrotic leaf spot in Phaseolus vulgaris at Puebla, Mexico

Beans are the most cultivated legume in the world. In Mexico, it is the second most important crop after corn (FAO 2020; SIAP 2020). Bean plants “Flor de Mayo M38” variety were affected by a foliar disease during the agricultural cycle 2019 in Puebla-Mexico (19°02'46.6" LN and 98°05'15.6" LO). Necrotic V- shaped lesions were observed on the margins of the leaves surrounded by yellow halos followed by foliar necrosis, affecting 40% of the crop. In Mexico this variety of cultivars is in great demand for local consumption and generates income in foreign currency (Castellanos et al. 1997). Sampling was carried out on 50 plants “Flor de Mayo M38” variety, with necrotic leaf symptoms from ten plots of one hectare. Samples were cut into pieces (5 mm), disinfested with 1% hypochlorite 3 min, and washed with sterile distilled water. Subsequently, samples were dried on sterile paper and placed on Petri plates containing yeast extract calcium carbonate dextrose agar (YDC) medium and kept at 36°C for 3 days. Colonies of ten typical bacteria isolated from all symptomatic plants were Gram (-), small and uniform in size with rounded edges, yellow, convex with entire borders and mucoid appearance on YDC. Bacteria did not grow on 0.1% triphenyl tetrazolium chloride amended casamino acid, peptone, and glucose medium (CPG). Biochemical tests showed that isolates did not reduce nitrate to nitrites, had positive catalase and starch hydrolysis, while the Kovac oxidase test was negative (Schaad and White 1974). Genus identity of the representative isolate Xcf1-APJR, was confirmed by 16S rRNA encoding gene partial sequencing, using universal primers 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5′-TACCAGGGTATCTAATCC-3′) (Halim et al. 2020). BLASTn alignments against the nucleotide collection were 100% identical to Xanthomonas sequences including Xanthomonas campestris pv. campestris strains NZ_AP019684.1, CP025750.1, and MN108237.1. The 1,418 bp sequence was deposited in the GenBank database under accession number MT645246. The identification of species/pathovar was accomplished by serological methods using a polyclonal antiserum specific for X. campestris pv. campestris (Popovic ́ et al. 2013) with the DAS-ELISA commercial kit (catalog number 07122C/096, LOEWE Biochemica GmbH, Germany). The pathogenicity test was carried out on 50 healthy bean plants from the "Flor de Mayo M38" variety. Bacterial culture incubated at 28°C for 48 h in YDC medium was used to prepare the bacterial suspension (108 CFU mL-1). The first two lower leaves of 30-day-old plants were inoculated by sprinkling. Ten plants sprayed with sterile distilled water were used as negative control. All plants were kept for 20 days in greenhouse at 18-26°C and relative humidity of 60%. After seven days, chlorotic lesions developed on all inoculated plants that became necrotic from 14 days after inoculation (dai). Necrotic leaf spots merged at 14 dai to form necrotic areas of more than 20 mm in diameter, reaching total necrosis of the leaf tissue at 20 dai and were similar to the symptoms observed in the field. Koch's postulates were confirmed by the reisolation of Xcf1-APJR strain, which presented the same colony morphology, partial sequence, and polyclonal specific detection. This is the first report of this pathogen causing necrotic leaf spot in beans from the "Flor de Mayo M38" variety in Puebla-Mexico. The author(s) declare no conflict of interest. References: FAO. 2020. FAOSTAT. Food and Agriculture Data. http://www.fao.org/faostat/en/#home/. SIAP. 2020. Atlas Agroalimentario. https://www.gob.mx/siap/. Castellanos, J. Z., et al. 1997. Arch. Latinoam. Nutr. 47:163. Schaad, N. W., and White, W. C. 1974. Phytopathology. 64:876. https://doi.org/10.1094/Phyto-64-876 Halim, R. A., et al. 2020. HAYATI J. Biosciences. 27:215. https://doi.org/10.4308/hjb.27.3.215 Popovic ́, T., et al. 2013. Plant Dis. 97:418. https://doi.org/10.1094/PDIS-05-12-0506-PDN

Genetic Diversity and Distribution of Korean Isolates of Burkholderia glumae

Burkholderia glumae causes panicle blight of rice (grain rot in Japan and Korea) and the severity of damage is increasing worldwide. During 2017‒2018, 137 isolates of B. glumae were isolated from symptomatic grain rot of rice cultivated in paddy fields throughout South Korea, and genetic diversity of the isolates was determined using transposase-based polymerase chain reaction (Tnp-PCR) genomic fingerprinting. All 138 isolates, including the B. glumae BGR1 strain, produced toxoflavin in various amounts, and 17 isolates produced an unidentified purple or orange pigment on Luria-Bertani (LB) and casamino acid-peptone-glucose (CPG) media, respectively, at 28°C. Tnp-PCR genomic fingerprinting was performed using a novel primer designed based on transposase (tnp) gene sequences located at the ends of the toxoflavin efflux transporter operon, and this method provided reliable and reproducible results. Through Tnp-PCR genomic fingerprinting, the genetic groups of Korean B. glumae isolates were divided into 11 clusters and three divisions. The Korean B. glumae isolates were mainly grouped in division I (73%). Interestingly, most of the pigment-producing isolates were grouped in divisions II and III, of which 10 were grouped in cluster VIII, which comprised 67% of this cluster. Phylogenetic analysis based on tofI and hrpB gene sequences was consistent with classification by Tnp-PCR genomic fingerprinting. The BGR1 strain did not belong to any of the clusters, indicating that this strain does not exhibit the typical genetic representation of B. glumae. Burkholderia glumae isolates showed diversity in the use of carbon and nitrogen sources, but no correlation with genetic classification by PCR fingerprinting was found. This is the first study to analyze the geographical distribution and genetic diversity of Korean B. glumae isolates.

Bacillus mojavensis: biofilm formation and biochemical investigation of its bioactive metabolites

Bacillus mojavensis is an endophytic bacterium which has been reported to have fungicidal effect against some phytopathogens. Bioactive secondary metabolites produced by B. mojavensis could have promising applications in agricultural, food industry and clinical fields. The current research has been conducted to: i) evaluate the antagonistic effect of B. mojavensis isolate against some phytopathogens; ii) characterize chemically the principal bioactive substances produced by the studied isolate of B. mojavensis using Gas Chromatography-Mass Spectroscopy (GC-MS); iii) evaluate its ability to produce a biofilm using ELISA technique. Results showed that the studied isolate has an antagonistic activity against the majority of tested microorganisms. Results showed also that the studied isolated produced a biofilm in Supplemented Luria-Bertani Casamino acid (SLB) and Minimal Mineral (MM) medium. The substantial attached growth in SLB was significantly higher than MM media. GC-MS analysis revealed the presence of 9 compounds accounting 87.8% of the total extract, where oxygenated monoterpenes are the main constituents.

EFFECT ON MICROBIAL PRODUCTS ON CAESIUM ELUTION BEHAVIOUR FROM CLAY MINERALS

Some microorganisms in the environment make siderophores, which are low molecular chelators, to take up minerals from soil. Eleven bacteria were separated from the root of white clover by chlome azrol S (CAS) assay. Each bacterium was incubated in casamino acid (CAA) culture, and siderophores in CAA culture were purified. These extractions were applied to biotite or vermiculite spiked with Cs. From each clay mineral, 57.1–72.8% (5100 ppm), 55.6–63.8% (920 ppm) and 48.6–54.3% (2300 ppm), 31.6–34.4% (520 ppm) was eluted, respectively. To understand elution behaviour, Cs desorption ratio of each clay was measured every 30 min. The results indicate Cs elution was occurred quickly.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum in Ghana, West Africa

Tomato and pepper plants exhibiting wilt symptoms were collected from fields in seven villages in Northern (Vea, Tono, Pwalugu), Ashanti (Agogo, Akumadan), and Brong Ahafo (Tanoso, Tuobodom) regions of western Ghana in November 2012. The plants were wilted without leaf yellowing or necrosis. Disease incidence was generally low, with less than 20% symptomatic plants observed. Most of the plants collected produced visible bacterial ooze in water in the field. Ooze was plated on 2,3,5-triphenyltetrazolium chloride-amended (TZC) medium. Isolated colonies were fluidal, irregularly round, white with pink centers, gram-negative, and oxidase positive. One strain from each of seven fields was selected for further study. All strains induced a hypersensitive reaction on tobacco. Randomly selected strains SM855-12 and SM857-12 tested positive in R. solanacearum ImmunoStrip assays (Agdia Inc., IN). An end-point PCR assay with primer set 759/760 (3) generated an R. solanacearum-specific 280-bp amplicon for all seven strains. Two of these strains were biovar I and the remaining five were biovar III based on utilization of cellobiose, lactose, maltose, dulcitol, mannitol, and sorbitol. A phylotype-specific multiplex PCR assay that recognizes four geographically linked monophyletic groups within R. solanacearum (1) indicated that one strain (SM855-12) was phylotype III (African origin), whereas the other six were phylotype I (Asian origin). All strains were subjected to repetitive sequence-based PCR (Rep-PCR) with BOXA1R and REP1R/REP2 primers (4). Strain SM855-12 was grouped with the phylotype III reference strain UW 368 and the remaining six strains were grouped with the phylotype I reference strain GMI 1000. A pathogenicity test was performed with bacterial wilt-susceptible tomato line OH7814. Inoculum was prepared from 48-h cultures of strains SM855-12, SM856-12, and SM858-12 grown on casamino acid peptone glucose (CPG) medium at 30°C. Roots of ten 4-week-old tomato plants per strain were drench-inoculated with 5 ml of a 108 CFU/ml bacterial suspension after wounding with a sterile scalpel. Non-inoculated control plants were drenched with 5 ml distilled water after root wounding. Plants were kept in a greenhouse at 25 to 30°C. By 12 days after inoculation, 80 to 100% of inoculated plants were wilted, whereas no symptoms appeared in non-inoculated plants. Bacteria re-isolated from wilted plants were confirmed to be R. solanacearum using techniques mentioned above. Although an association of bacterial wilt with tomato/pepper was mentioned previously (2), to our knowledge, this is the first documented report of bacterial wilt caused by R. solanacearum in Ghana. The presence of Asian strains (phylotype I) may be the result of one or more accidental introductions. Awareness of this disease in Ghana will lead to deployment of management strategies including use of resistant varieties and grafting desirable varieties onto disease-resistant rootstocks. References: (1) M. Fegan and P. Prior. Page 449 in Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. C. Allen et al., eds. American Phytopathological Society, St. Paul, MN, 2005. (2) K. A. Oduro. Plant Protection and Regulatory Services Directorate of MOFA, Accra, Ghana, 2000. (3) N. Opina et al. Asia Pac. J. Mol. Biol. Biotechnol. 5:19, 1977. (4) J. Versalovic et al. Methods Mol. Cell Biol. 5:25, 1994.

Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Ağri, Turkey

A novel amylolytic enzyme producing thermophilic bacterial strain KP1 from the Diyadin hot spring water in Ağri, Turkey, was isolated in the present study. Phylogenetic analysis based on the partial 16S rRNA gene, biochemical and physiological tests revealed that the strain KP1 belongs to the genus Anoxybacillus. The pH and temperature optima for the α-amylase production by Anoxybacillus sp. KP1 were 8.0 and 50°C, respectively, where the maximum growth was obtained at the 28th hour of incubation and the highest α-amylase activity was obtained at the 40th hour of incubation (8979.6 U/mL). The optimum pH and temperature for the enzyme activity were 8.0 and 60°C, respectively. The maximum α-amylase production was secreted in the presence of 2% (w/v) soluble starch (10837.7 U/mL). Among the various organic and inorganic nitrogen sources tested, while keeping the beef extract concentration constant, casamino acid (14310.6 U/mL), urea (14126 U/mL), and tryptone (13217.2 U/mL) at a concentration of 2% gave the maximum α-amylase production. The enzyme activity was enhanced in the presence of 1.5 mM Mn2+ (123%), whereas it was strongly inhibited 1.5 mM by Hg2+. Inhibition by 89% was obtained also with sodium dodecyl sulphate (1%). The enzyme was found to be relatively stable at a range of pH and temperature.

Improvement of cyclodextrin glycosyltransferase (CGTase) production by recombinant Escherichia coli pAD26 immobilized on the cotton

The cyclodextrin glycosyltransferase (CGTase) of the recombinants Escherichia coli pAD26 cells immobilized on cotton was optimally produced by statistical methodology. Primarily, carbon and nitrogen sources were selected by one-factor-at-a-time method. Wheat starch, Casamino acid, Edamin and Hy-soy were identified as the best nutrients. These sources were secondly confirmed by Plackett-Burman design (fifteen variables were studied with sixteen experiments), as the most significant components with respect to CGTase production. In the third step, concentration of most significant factors and their interaction were optimized with a Box-Behnken experimental design. Under the optimized conditions (agitation 200 rpm, yeast extract concentration 20 g/L, wheat starch concentration 10 g/L and Hy-soy concentration 2.5 g/L), CGTase yield 145.11 U/mL was 3.6 and 23 folds higher than those obtained by the use of the initial conditions (39.77 U/mL) and free cells (6.37 U/mL), respectively.

Leaf Stripe and Stem Rot Caused by Burkholderia gladioli, a New Disease of Maize in México

A new maize disease appeared in the State of Veracruz, Mexico during 2003–2004. Initial symptoms in the leaves were small, white-yellow, watery spots, which coalesced into dry necrotic stripes that were 0.3 wide and 8 cm long. Reddening sometimes developed on these leaves. Stems developed a rot in the crown. The flag leaf showed a rot and necrosis at the base, rolled inward, and dried out. Necrosis developed at the base of the corn ears and their growth was halted. A bacterium characterized by white colonies was consistently isolated from lesions on casamino acid peptone and glucose (CPG), King's medium B, and nutrient agar media. Ten isolates were chosen for further characterization. Pathogenicity was confirmed in the greenhouse (25 to 30°C) on 45-day-old ASGROW 7573 maize plants by injection of bacterial suspensions (107 CFU/ml) at the base of the stem. Control maize seedlings were injected with phosphate buffer. Symptoms similar to those observed in the field were observed after 3 days on all inoculated plants but were not observed on control plants. Koch's postulates were fulfilled with reisolation of the inoculated strain from the inoculated tissues and confirmation was fulfilled by phenotypic characters and 16S rDNA sequences. The white colonies on CPG were slightly convex, shiny, circular with entire margins, gram negative, lacked arginine dihydrolase, did not produce fluorescent pigment on Pseudomonas F medium, and grew aerobically. The strains were able to utilize l-arabinose, d-mannitol, and cellobiose, but unable to utilize d-maltose and l-rhamnose. Gel hydrolysis was positive but starch hydrolysis was not positive. Colonies grew at 40°C. These characteristics are the same as those described previously for Burkholderia gladioli (3). The identity of these isolates was confirmed by 16S analysis with two universal primers, FD1 and RD1, for Eubacteria (2), which generate a 1,600-bp fragment. Two primers specific for the genus Burhkolderia, RHG-F and RHG-R that generate a 500-bp fragment (1) were also used for amplification. Comparison of sequences using Clustal W and Megaline, DNAstar software showed 100% similarity among strains isolated from the three counties (GenBank Accession Nos. EU161873 to EU161878) and 99% similarity of field isolates with B. gladioli in the GenBank database. To our knowledge, this is the first report of B. gladioli causing leaf stripe and stem rot of maize in Mexico. References: (1) J. J. Lipuma et al. J. Clin. Microbiol. 37:3167, 1999. (2) L. M. Rodrigues et al. Appl. Environ. Microbiol. 69:4249, 2003. 3) N. W. Shaad et al., eds. 3rd ed. Laboratory Guide for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN, 2001.

Structure Proposal for a New Pyoverdin from Pseudomonas sp. PS 6.10

From Pseudomonas sp. PS 6.10, when grown in a casamino acid medium, a pyoverdin was isolated whose primary structure could be deduced from its mass spectrometric fragmentation pattern and amino acid analysis. It belongs to the smallest representatives of this group of siderophores comprising only six amino acids in its peptide chain. When grown in a succinate minimal medium the corresponding ferribactin considered to be the biogenetic precursor of the pyoverdin was obtained as the major component.