First Report of Bacterial Canker on Blueberry (Vaccinium corymbosum) Caused by Pseudomonas syringae pv. syringae in Serbia

During May 2021, necrosis of young twigs and flower buds were observed on two-year-old highbush blueberry plants (Vaccinium corymbosum) cv. Draper, in a 1 hectare orchard in the municipality of Šabac, Serbia. Disease symptoms included reddish-brown to black irregularly shaped cankers developing on the shoot tips that extended downwards along the branches. In some plants, cankers surrounded the stem, causing shoot-tip dieback and necrosis of the buds. Beneath the bark, a distinct margin between diseased and healthy tissue was visible. A few weeks before symptoms development, seven freezing events with temperature from -3°C to -1°C, and five near-freezing temperatures were recorded in this area, leading to the hypothesis that symptoms were associated to the presence of ice nucleating bacteria belonging to Pseudomonas syringae. The observed disease incidence was 80%, while 10% of the plants died. Bacteria were isolated from symptomatic tissue on King’s medium B (KB). After 2 to 3 days of incubation at 27°C, predominantly grey-whitish, shiny, round, convex bacterial colonies were observed on agar plates. Ten isolates producing a fluorescent pigment on KB were selected for further characterization by biochemical and molecular tests. The isolates were Gram, oxidase and arginine-dihydrolase negative, levan positive, induced hypersensitive response on tobacco leaves and showed no pectinolytic activity on potato slices. Based on the results of API 20E and API 20NE tests (BioMerieux, France), and the fact that isolates did not utilize tartrate nor had tyrosinase activity, they were preliminarly identified as Pseudomonas syrinage pv. syringae (Braun-Kiewnick and Sands 2001). Additionally, all tested isolates had ice-nucleation activity at -5°C. The syrB gene responsible for syringomycin synthesis, was amplified in all isoaltes with the specific primer pair B1/B2 (Sorensen et al. 1998). The 16S rRNA gene sequences of five selected isolates (GenBank MZ410287 to 91) showed 100% identity to P. s. pv. syringae isolated from Prunus avium in United Kingdom (GenBank CP026568) and France (GenBank LT962480). Sequences of gyrB gene (Sarkar and Guttman 2004) of two selected isolates (GenBank MZ420633 and MZ420634) showed 98,44% identity to the P. s. pv. syrinage strain isolated in France (GenBank LT962480). Pathogenicity of the isolates was confirmed on 2-year-old blueberry plants cv. Draper, by inoculating two plants per isolate. One-cm long wounds were made on branches using a scalpel and 20 µl of bacterial suspension (106 CFU/ml) was infiltrated into the tissue. The cuts were then covered with moist sterile cotton pads and wraped in parafilm for 3 days. Inoculation was also performed on two leaves per plant by needleless syringe infiltration (106 CFU/ml). Sterile distilled water was used as a negative control. Plants were maintained in greenhouse at 27°C day and 15°C night temperature. Three weeks after inoculation, the inoculated branches and leaves developed necrosis, leaves spots and cankers respectively, resembling the natural infection. Symptoms were not observed on the control plants. Bacteria were reisolated from symptomatic tissue and their identity was confirmed by amplifying the syrB gene sequence and additional biochemical tests. This is the first report of bacterial canker of highbush blueberry caused by P. s. pv. syringae in Serbia. In Europe, there was only one report on Pseudomonas spp. causing disease on blueberry leaves in Poland (Kaluzna et al. 2013). Due to market demands and export potential, blueberry production in Serbia has been rapidly increasing. In 2015, highbush blueberry was cultivated on 220 ha, while in 2020 the area increased to 1899 ha. However, under favourable environmental conditions, blueberry production might be severely affected by bacterial canker. References: Braun-Kiewnick, A. and Sands, D.C. Page 84 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. Kaluzna, M., et al. 2013. J Plant Protec Res 53:32. Sarkar, S. F., and Guttman, D. S. 2004. Appl. Environ. Microbiol. 70:1999. https://doi.org/10.1128 Sorensen, K. N., et al. 1998. Appl. Environ. Microbiol. 64:226.

mSphere of Influence: Turning to Soil for Medicines

ABSTRACT Ching-Hsuan Lin works in the field of Candida biology. In this mSphere of Influence article, he reflects on how the papers “Use of ichip for high-throughput in situ cultivation of uncultivable microbial species” by D. Nichols, N. Cahoon, E. M. Trakhtenberg, L. Pham, et al. (Appl Environ Microbiol 76:2445–2450, 2010, https://doi.org/10.1128/AEM.01754-09) and “A new antibiotic kills pathogens without detectable resistance” by L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, et al. (Nature 517:455–459, 2015, https://doi.org/10.1038/nature14098) made an impact on him by inspiring him to explore new bioactive antimicrobial compounds with his collaborators.

Оптимизация методики получения рекомбинантных двухдоменных лакказ

Лакказы (К.Ф. 1.10.3.2) – ферменты из семейства медьсодержащих оксидаз, активный центркоторых содержит 4 атома меди. Лакказы способны окислять широкий спектр органический и неорганических соединений. Ферменты данного класса используют в биотехнологических целях (в целлюлозно-бумажной, текстильной, пищевой промышленности). В структурном отношении лакказыподразделяют на 2 группы: двухдоменные (2д) и трёхдоменные (3д) ферменты. Характерные особенности 2д лакказ – устойчивость к специфичным ингибиторам семейства медьсодержащих оксидаз, а также высокая термостабильность. Окислительно-восстановительный потенциал 2д лакказ ниже потенциала 3д ферментов. Однако он может быть повышен благодаря использованию редоксмедиаторов. Высокая термостабильность и устойчивость к действию ингибиторов – важные критерии отбора ферментов для нужд биотехнологии. Также важным критерием для биотехнологически значимых ферментов является стоимость их производства. Если получение фермента требует значительных затрат, а выход конечного продукта низок, то производство фермента нецелесообразно.Поэтому целью данной работы является оптимизация процесса получения двухдоменных рекомбинантных лакказ, экспрессируемых гетерологично в штамме Escherichia coli, с расчетами стоимости конечного продукта (на примере ферментов SgfSL, SvSL и SaSL, полученных в нашей лаборатории). Ранее три рекомбинантные двухдоменные лакказы были клонированы и экспрессированы в штамме Escherichia coli M15 (pRep4). В данной работе мы исследовали влияние различных факторов на максимальный выход лакказ: влияние ионов меди, концентрации индуктора, условий культивирования, оптической плотности культуры, и других условий. Было показано, что оптимальная концентрация ионов меди составляет 1 мМ, а оптимальная концентрация индуктора ИПТГ составляет 0,1мМ (при этом отсутствует эффект агрегирования и наблюдается высокий выход ферментов). Мы подтвердили выводы коллег о том, что для получения лакказ, максимально насыщенных ионами меди, необходимы микроаэробные условия культивирования. Без стадии микроаэробного роста удельная активность очищенных ферментов снижается в 2 раза. Было обнаружено, что слишком высокая скорость перемешивания клеток при индукции синтеза лакказ приводит к агрегации ферментов. Скорость перемешивания, при которой лакказы не агрегируют, составляет 50-100 об/мин.Выводы: был разработан и оптимизирован процесс получения двухдоменных бактериальныхрекомбинантных лакказ. Максимальный выход ферментов составил 180 мг белка с литра среды. Фер-мент имел низкую себестоимость (16-32 евро за 1 г белка). ЛИТЕРАТУРА 1. Baldrian P. // FEMS Microbiol Lett. 2006. Vol. 30. No 2. pp. 215-242.2. Claus H. // Arch Microbiol. 2003. Vol. 179. No 3. pp. 145-150.3. Otto B., Schlosser D. // Planta. 2014. Vol. 240. No 6. pp. 1225-1236.4. Lisov A.V., Zavarzina A.G., Zavarzin A.A., Leontievsky A.A. // FEMS Microbiol Lett.2007. Vol. 275. No 1. pp. 46-52.5. Thurston C.F. // Microbiology. 1994. Vol. 140. pp. 19-26.6. Sterjiades R., Dean J.F., Eriksson K.E. // Plant Physiol. 1992. Vol. 99. No 3. pp. 1162-1168.7. Endo K., Hosono K., Beppu T., Ueda K. // Microbiology. 2002. Vol. 148. pp. 1767-1776.8. Lu L., Zeng G., Fan C., Zhang J. et al. // Appl Environ Microbiol. 2014. Vol. 80. No 11. pp. 3305-3314.9. Minussi R.C., Pastore G.M., Duran N. // Trends Food Sci Tech. 2002. Vol. 13. No 6-7. рр. 205-216.10. Dominguez A., Couto S.R., Sanroman M.A. // World J Microbiol Biotechnol. 2005. Vol. 21. No 4. pp. 405-409.11. Couto S.R., Herrera J.L.T. // Biotechnol Adv. 2006. Vol. 24. No 5. pp. 500-513.12. Trubitsina L.I., Tishchenko S.V., Gabdulkhakov A.G., Lisov A.V. et al. // Biochimie.2015. Vol. 112. pp. 151-159.13. Tishchenko S., Gabdulkhakov A., Trubitsina L., Lisov A. et al. // Acta Crystallogr F Struct Biol Commun. 2015. Vol. 71. pp. 1200-1204.14. Lisov A.V., Trubitsina L.I., Lisova Z.A., Trubitsin I.V. et al. // Process biochemistry. 2019. Vol. 76. pp. 128-135.15. Durao P., Chen Z., Fernandes A.T., Hildebrandt P. et al. // J Biol Inorg Chem. 2008. Vol. 13. No 2. pp. 183-193.16. Gunne M., Urlacher V.B. // PLoS One. 2012. Vol. 7. No 12. pp. e52360.

The Ability of Ginger Rhizome (Zingiber officinale Rosc) Extract in Producing of Silver Nanoparticles and the Antibacterial activity of these nanoparticles

Recently, using plant extract as a reducing agent for nanosilver particle synthesis hasbeen focused. This is a green technology utilizing the ready material in the nature to create thenanoparticles with good properties and uniqe quality. In this study, ginger rhizome extract wasused to reduce the silver cation (Ag + ) to silver (Ag o ) as nanoparticles with uniqe quality and evendistribution in the solution. The size of the particles varied in the range of 20-40 nm. Reactionconditions were investigated and optimized with AgNO 3 concentration of 3mM, extractsolution/AgNO 3 solution of 1/5, temperature of 80˚C, pH of 12 and reaction time of 30 min. Theresults obtained from the antibacterial assays showed that silver nanoparticle solution hadantibacterial ability with an average effective diameter of 10 mm. It also indicated that theantibacterial activity of silver nanoparticle solution on the Gram (-) bacterium (E. coli) is betterthat on Gram (+) bacterium (S. aureus). In conclusion, we suggest that the ginger rhizome extractcan be used to produce silver nanoparticles in mild reaction conditions; the silver nanoparticlesolution expressed as a quite good antibacterial agent and therefore could be applied in decreasingthe effects of deleterious bacteria.Keywords Silver nanoparticle, plant extract, antibacterial, Zingiber officinale Rosc. References [1] L.S. Li, J. Hu, W. Yang and P. Alivisatos. Band gap variation of size- and shape-controlled colloidal CdSe quantum rods. Nano Lett. 1(2001) 49-51. https://doi.org/10.1021/nl015559r.[2] A. P. Nikalje. Nanotechnology and its Applications in Medicine. Medicinal chemistry, 5(2015) 81-89.[3] G. Doria, J. Conde, B. Veigas et al. Noble metal nanoparticles for biosensing applications. Sensors 12(2012) 1657–1687. https://doi.org/ 10.4172/2161 -0444.1000247[4] A. J. Haes, A. D. McFarlan, R. P. van Duyne. Nanoparticle optics: sensing with nanoparticle arrays and single nanoparticles. The International Society for Optical Engineering 5223 (2003) 197–207. https://doi.org/10.1039/C7NR03311G.[5] A. Elham, M. Morteza, F. V. Sedigheh, K. Mohammad, A. Abolfazl, T. N. Hamid, N. Parisa, W. J. San, H. Younes, N-K. Kazem, S. Mohammad. Silver nanoparticcles: Synthesis methods, bio-applications and properties. Critical reviews in Microbiology 42(2016) 173-180. https://doi.org/10.3109/1040841X.2014.912200.[6] J. K. Pradeep, K. Chaudhury, V. S. Suresh, K. G. Sujoy. An emerging interface between life science and nanotechnology: present status and prospects of reproductive healthcare aided by nano-biotechnology. Nano Rev. 5(2014): 10.3402/ nano. v5. 22762. https://doi.org/10.3402/nano.v5.22762.[7] M. Danilcauk, A. Lund, J. Saldo, H. Yamada, J. Michalik. Conduction electron spin resonance of small silver particles. Spectrochimaca. Acta. Part A 63(2006) 189–191. https://doi.org/10.1016/j.saa. 2005.05.002[8] J. L. Elechiguerra, J. L. Burt, J. R. Morones et al. Interaction of silver nanoparticles with HIV-1. Journal of Nanobiotechnology 3(2005) 6. https:// doi.org/10.1186/1477-3155-3-6[9] J. S. Kim, E. Kuk, K. Yu, J. H. Kim, S. J. Park, H. J. Lee, S. H. Kim, Y. K. Park, Y. H. Park, C. Y. Hwang, Y. K. Kim, Y. S. Lee, D. H. Jeong, M. H. Cho. Antimicrobial effects of silver nanoparticles. Nanomedicine 3(2007) 95–101. https://doi.org/ 10.1016/j.nano.2006.12.001.[10] Y. Matsumura, K. Yoshikata, S. Kunisaki and T. Tsuchido. Mode of bacterial action of silver zeolite and its comparison with that of silver nitrate. Appl. Environ. Microbiol. 69(2003) 4278–4281.https://doi.org/10.1128/AEM.69.7.4278-4281. 2003.[11] M. Yamanaka, K. Hara, J. Kudo. Bactericidal Actions of a Silver Ion Solution on Escherichia coli, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis. Appl. Environ. Microbiol. 71(2005) 7589–7593. https://doi.org/10.1128/AEM.71.11.7589-7593. 2005.[12] Y. H. Hsueh, K. S. Lin, W. J. Ke, C. T. Hsieh, C. L. Chiang, D. Y. Tzou and S. T. Liu. The Antimicrobial Properties of Silver Nanoparticles in Bacillus subtilis Are Mediated by Released Ag+ Ions. PLoS One 10(2015):e0144306. https://doi.org/10.1371/journal.pone.0144306.[13] N. Kumar, S. Das, A. Jyoti and S. Kaushik. Synergistic effect of silver nanoparticles with doxycycline against Klebsiella pneumoniae. Int. J. Pharm. Sci. 8(2016) 183-186.[14] V. G. Borodina, Y. A. Mirgorod. Kinetics and Mechanism of Interaction between HAuCl4 and Rutin. Kinet. Cat. 55(2014) 683–687. https://doi. org/10.1134/S0023158414060044.[15] V. V. Makarov, A. J. Love, O. V. Sinitsyna, S. S. Makarova, I. V. Yaminsky, M. E. Taliansky, N. O. Kalinina. Green nanotechnologies: synthesis of metal nanoparticles using plants. Acta Naturae 6(2014) 35–44. https://doi.org/10.1039/C1GC15 386B. [16] M. S. Butt, M. T. Sultan. Ginger and its health claims: molecular aspects. Critical Reviews in Food Science and Nutrition 51(2011) 383–393. https://doi.org/10.1080/10408391003624848[17] M. Park, J. Bae, D. S. Lee. Antibacterial activity of gingerol and gingerol isolated from ginger rhizome against periodontal bacterial. Phytotherapy Research 22(2008) 1446–1449. https://doi.org/10.1002/ptr.2473[18] Y. Shukla, M. Singh. Cancer preventive properties of ginger: a brief review. Food and Chemical Toxicology 45(2007) 683–690. https://doi.org/10. 1016/j.fct.2006.11.002.

First Report of Fire Blight on Pear Trees Caused by Erwinia amylovora in Kurdistan Province, Iran

Pear (Pyrus L.) is one of the most widely grown crops in western Iran. Since 2010, an outbreak of a disease with symptoms similar to fire blight has been observed on pear trees in various locations of Kurdistan Province. Initial flower symptoms include water-soaking and rapidly shriveling, infected flowers that remained hanging on the trees. Immature fruits become water-soaked, turned brown, and shriveled. Infected flowers and immature fruits were collected from different locations in the province. Small pieces (about 1 mm2) were excised from infected tissues, surface sterilized with 0.5% sodium hypochlorite solution, followed by rinsing in sterile-distilled water (SDW). Each piece was macerated in 2 to 3 ml of SDW, streaked onto nutrient agar sucrose or eosin methylene blue agar media, and incubated at 27 to 29°C. After 48 to 72 h, single colonies were subcultured onto the same media and stored at 4°C. In total, 74 bacteria were isolated from infected tissues. All isolates were gram-negative and rod-shaped. Based on other phenotypic properties, strains were grouped into three clusters at a similarity level of 65% (data not shown). Forty-one and 23 strains showed properties as expected for Erwinia amylovora and Enterobacter sp., respectively. Other strains showed properties resembling Pantoea agglomerans. All strains identified as E. amylovora produced an expected DNA fragment of about 900 bp by PCR using primers PE29A and PE29B corresponding to plasmid pEA29 (1). The result was confirmed by using primers AMSbL and AMSbR derived from the ams region required for amylovoran synthesis of E. amylovora. E. amylovora strains produced an expected 1,600-bp fragment (2). For the pathogenicity test, a bacterial suspension was adjusted to approximately 1 × 107 CFU/ml from cell cultures grown in nutrient broth at 27°C for 48 h. Immature pear fruits sterilized with 70% ethanol and rinsed with SDW were injected with the bacterial suspension using a 25-gauge sterile needle. Fruits injected with sterile water were used as controls. Pear fruits were kept in a mist chamber at 27 to 29°C. Symptoms were assessed up to 2 weeks after inoculation. All E. amylovora strains produced typical symptoms on inoculated immature pear fruits. Necrosis and oozing of bacterial exudates were observed after 3 to 7 days. The phylogenetic position of two selected strains was analyzed by sequence comparison of recA gene among other species in the genus Erwinia and related bacteria. The recA sequence of bacterial strains identified as E. amylovora revealed high similarity (99%) to the E. amylovora type strain (CFBP 1430). Genetic diversity of selected strains was assessed and compared with E. amylovora reference strain CFBP 1430 using ERIC and REP primers in rep-PCR analysis. (3). UPGMA cluster analysis of the combined data obtained in the rep-PCR experiments using Dice's coefficient revealed that the majority of E. amylovora strains showed the same fingerprint patterns at a similarity level of 93%, indicating genetic homogeneity among strains but clearly separated from Enterobacter sp. and P. agglomerans strains. To our knowledge, this is the first report that characterizes the phenotypic and genetic properties of E. amylovora in western part of Iran. References: (1) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992. (2) S. Bereswill et al. Appl. Environ. Microbiol. 61:2636, 1995. (3) J. Versalovic et al. Mol. Cell Biol. 5:25, 1994.

First Report of Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum on Pepper Fruits (Capsicum annuum) in Malaysia

Symptoms of water-soaked lesions and soft rot were first observed in June 2011 on bell pepper fruits (Capsicum annuum cv. Annuum) in the two main regions of pepper production in Malaysia (Cameron Highlands and Johor State). Economic losses exceeded 40% in severely infected fields and greenhouses with the estimated disease incidence of 70%. In pepper fruits damaged by insects, sunscald, or other factors, symptoms initially appeared in the peduncle and calyx tissues and entire fruits were turned into watery masses within 2 to 6 days. Fruits infected in the field tended to collapse and hang on the plant. When the contents leaked out, the outer skin of the fruit dried and remained attached to the plant. Field-grown transplants and infected soil were identified as probable sources of inocula. A total of 50 attached fruits were collected from 10 pepper fields and greenhouses located in the two growing regions. Tissue from the margins of water-soaked lesions was surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto nutrient agar (NA) and eosin methylene blue agar (EMB) media (3). A similar bacterium was isolated from all samples. After 2 days, white to creamy bacterial colonies on NA and emerald green colonies on EMB developed. Five independent strains were subjected to further biochemical, molecular, and pathogenicity tests. Bacterial strains were gram-negative, motile rods, grew at 37°C, were facultatively anaerobic, oxidase-negative, phosphatase-negative, and catalase-positive. They degraded pectate, were sensitive to erythromycin, did not utilize Keto-methyl glucoside, were indole production-negative, and reduced sugars from sucrose (3). Acid production was negative from sorbitol and arabitol, but positive from melibiose and citrate. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment (2). Amplification of the intergenic transcribed spacer (ITS) region by G1 and L1 primers (4) gave two amplicons ca. 550 and 580 bp long. The expected amplicon was not produced with any of the strains using primers Br1f/L1r and Eca1f/Eca2r (1), whereas a 550-bp PCR product, typical of Pectobacterium carotovorum subsp. carotovorum, was obtained with primers EXPCCF and EXPCCR (1). Based on biochemical and molecular characteristics, and analysis of PCR-RFLP of 16S-ITS-23R rRNA genes using Rsa I enzyme (4), all five bacterial strains were identified as P. carotovorum subsp. carotovorum. BLAST analysis of the 16S rRNA sequence (GenBank Accession No KC189032) showed 100% identity to the 16S rRNA of P. carotovorum subsp. carotovorum strain PPC192. For pathogenicity tests, four mature pepper fruits of cv. Annuum were inoculated by injecting 10 μl of a bacterial suspension (108 CFU/ml) into pericarps and the fruits were incubated in a moist chamber at 80 to 90% relative humidity and 30°C. After 72 h, water-soaked lesions similar to those observed in the fields and greenhouses were observed and bacteria with the same characteristics were consistently reisolated, thereby fulfilling Koch's postulates. Symptoms were not observed on water-inoculated controls. References: (1) S. Baghaee-Ravari et al. Eur. J. Plant Pathol. 129:413, 2001. (2) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (3) N. W Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society Press, St Paul, MN, 2001. (4) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.

First Report of Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum on Cucumber in Malaysia

Cucumber (Cucumis sativus L.) is one of the most important vegetable fruits in Malaysia. Cucumber is principally grown in the states of Johor, Kelantan, and Perak. The broad host range Enterobacteriaceae pathogen, Pectobacterium carotovorum, can cause soft rot on stems or cucumber fruit. In Malaysia, cucumber is produced in a warm, humid climate, thus the plant is susceptible to attack by P. carotovorum at any time during production. In 2010, cucumber samples with wilted and chlorotic leaves, water-soaked lesions, and collapsed fruits were found in multiple fields. Small pieces of infected stems and fruit were immersed in 5 ml of saline solution (0.85% NaCl) for 20 min and then 50 μl of this suspension was spread onto nutrient agar (NA) and incubated at 27°C for 24 h. White-to-pale gray colonies with irregular margins were selected for analysis. For pathogenicity tests, cucumber fruits were surface sterilized by ethyl alcohol 70%, washed with sterilized distilled water, cut into small pieces, and inoculated with 20 μl of 108 CFU/ml suspensions of five representative strains. Cucumber plants were grown for 3 weeks in sterilized soil and their stems were inoculated with 20 μl of 108 CFU/ml of bacterial suspension. Inoculated samples and control (noninoculated) plants were placed in a growth chamber with 80 to 90% relative humidity at 27°C. Symptoms occurred on fruit slices and stems after 1 to 3 days and appeared the same as naturally infected samples, but the control samples remained healthy. Koch's postulates were fulfilled with the reisolation of cultures with the same characteristics as described earlier. Hypersensitivity reaction (HR) assays were done by infiltrating 108 CFU/ml of bacterial suspension into tobacco leaf epidermis and HR developed. All strains were subjected to biochemical and morphological assays, as well as molecular assessment. The strains were gram negative, facultative anaerobes, rod shaped, able to macerate potato slices and growth at 37°C; catalase positive; oxidase and phosphatase negative; able to degrade pectate; sensitive to erythromycin; negative for utilization of α-methyl glycoside, indole production, and reduction of sugars from sucrose; acid production from arabitol, sorbitol, and utilization of citrate were negative, but positive for raffinose and melibiose utilization. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment on agarose gel 1% (1). Amplification of intergenic transcribed spacer region by G1 and L1 primers gave two main bands at approximately 535 and 580 bp on agarose gel 1.5%. The ITS-PCR products were digested with RsaI restriction enzyme (3). On the basis of biochemical and morphological characteristics, PCR-based pel gene and characterization of the ITS region, and digestion of the ITS-PCR products with RsaI restriction enzyme, all isolates were identified as P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of soft rot caused by P. carotovorum subsp. carotovorum on cucumber from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society Press, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.

First Report of Cabbage Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Malaysia

Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetables cultivated in Pahang and Kelantan, Malaysia. Pectobacterium carotovorum can cause soft rot on a wide range of crops worldwide, especially in countries with warm and humid climates such as Malaysia. Cabbage with symptoms of soft rot from commercial fields were sampled and brought to the laboratory during the winter of 2010. Disease symptoms were a gray to pale brown discoloration and expanding water-soaked lesions on leaves. Several cabbage fields producing white cultivars were investigated and 27 samples were collected. Small pieces of leaf samples were immersed in 5 ml of saline solution (0.80% NaCl) for 20 min to disperse the bacterial cells. Fifty microliters of the resulting suspension was spread on nutrient agar (NA) and King's B medium and incubated at 30°C for 48 h. Purification of cultures was repeated twice on these media. Biochemical and phenotypical tests gave these results: gram negative, rod shaped, ability to grow under liquid paraffin (facultative anaerobe); oxidase negative; phosphatase negative; positive degradation of pectate; sensitive to erythromycin; negative to Keto-methyl glucoside utilization, indole production and reduction sugars from sucrose were negative; acid production from sorbitol and arabitol was negative and from melibiose, citrate, and raffinose was positive. Hypersensitivity reaction on tobacco leaf with the injection of 106 CFU/ml of bacterial suspension for all strains was positive. Four representative strains were able to cause soft rot using cabbage slices (three replications) inoculated with a bacterial suspension at 106 CFU/ml. Inoculated cabbage slices were incubated in a moist chamber at 80% relative humidity and disease symptoms occurred after 24 h. Cabbage slices inoculated with water as a control remained healthy. The bacteria reisolated from rotted cabbage slices on NA had P. carotovorum cultural characteristics and could cause soft rot in subsequent tests. PCR amplification with Y1 and Y2 primers (1), which are specific for P. carotovorum, produced a 434-bp band with 15 strains. PCR amplification of the 16S-23S rRNA intergenic transcribed spacer region (ITS) using G1 and L1 primers gave two main bands approximately 535 and 580 bp and one faint band approximately 740 bp when electrophoresed through a 1.5% agarose gel. The ITS-PCR products were digested with RsaI restriction enzyme. According to biochemical and physiological characterictics (2), PCR-based pel gene (1), and analysis by ITS-PCR and ITS-restriction fragment length polymorphism (3), all isolates were identified as P. carotovorum subsp. carotovorum. This pathogen has been reported from Thailand, Indonesia, and Singapore with whom Malaysia shares its boundaries. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum in cabbage from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W. Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.

Cadherin Fragments from Anopheles gambiae Synergize Bacillus thuringiensis Cry4Ba's Toxicity against Aedes aegypti Larvae

ABSTRACT A peptide from cadherin AgCad1 of Anopheles gambiae larvae was reported as a synergist of Bacillus thuringiensis subsp. israelensis Cry4Ba's toxicity to the Anopheles mosquito (G. Hua, R. Zhang, M. A. Abdullah, and M. J. Adang, Biochemistry 47:5101-5110, 2008). We report that CR11 to the membrane proximal extracellular domain (MPED) (CR11-MPED) and a longer peptide, CR9 to CR11 (CR9-11), from AgCad1 act as synergists of Cry4Ba's toxicity to Aedes aegypti larvae, but a Diabrotica virgifera virgifera cadherin-based synergist of Cry3 (Y. Park, M. A. F. Abdullah, M. D. Taylor, K. Rahman, and M. J. Adang, Appl. Environ. Microbiol. 75:3086-3092, 2009) did not affect Cry4Ba's toxicity. Peptides CR9-11 and CR11-MPED bound Cry4Ba with high affinity (13 nM and 23 nM, respectively) and inhibited Cry4Ba binding to the larval A. aegypti brush border membrane. The longer CR9-11 fragment was more potent than CR11-MPED in enhancing Cry4Ba against A. aegypti.