First Report of bacterial wilt disease of banana caused by Klebsiella variicola in Malaysia.

In Malaysia, bananas (Musa spp.) are the second most cultivated fruit and the fourth most cultivated fruit in terms of export revenue. In October 2018, about 5.0 out of 6.6 hectares of a banana plantation located in Teluk Intan, Malaysia, was impacted by an outbreak of banana disease. The onset of bacterial wilt symptoms is characterized by initial leaf wilting, followed by the subsequent withering of the entire plant during later stages, fruit stalk and fruit pulp discoloration, fruit rotting, and pseudostem necrosis. The diseased banana's symptomatic pseudostems and fruit pulps were surface-sterilised in 70% ethanol for 30 s, followed by 2% NaClO for 3 min, rinsed three times in sterilised water, and cut into small pieces approximately 5 mm2 in size. The tissues were macerated in a sterilised 0.85% NaCl solution for 5 min, and the resulting suspension was streaked onto nutrient agar, followed by incubation at 28°C for 2 days. After incubation, bacterial colonies with five unique morphological characteristics were observed. Two colonies of each unique morphological type were randomly chosen and subjected to preliminary bacterial identification by 16S rRNA gene sequencing. Based on BLASTn analysis, the five unique morphological types of bacteria were preliminarily identified as Enterobacter cloacae, Citrobacter farmeri, Klebsiella variicola, Kosakonia radicincitans, and Phytobacter ursingii. Previous reports identified K. variicola and K. radicincitans as banana pathogens, but Malaysia has yet to report the former. The amplified partial 16S rDNA sequences of both K. variicola isolates (designated as UTAR-BC1 and UTAR-BC2; GenBank accession numbers: PP531448 and PP531460, respectively), which were chosen to be the focus of this study, exhibited complete similarity to each other and were 100% identical (1426/1426 identity and 1420/1420 identity, respectively) to K. variicola (CP026013.1). To verify the identity of the bacterial isolate, three housekeeping genes, namely, infB(PP538994), rpoB (PP538995), and gyrB (PP538996) of UTAR-BC1, were amplified, sequenced, and subjected to multilocus phylogenetic analysis via the neighbour-joining method (1,000 bootstrap values). Phylogenetic analysis revealed that UTAR-BC1 belongs to the K. variicola clade. A pathogenicity assay of UTAR-BC1 was conducted on 4-month-old healthy banana plantlets (cv. Nangka) using the pseudostem injection method (Tripathi et al., 2008). First, UTAR-BC1 was grown overnight in nutrient broth and then adjusted to 108 CFU/ml in a sterile 10 mM MgCl2 solution. A total volume of 100 µL of the bacterial suspension was injected into the pseudostem of five healthy banana plantlets via a syringe with a needle. Control plants were mock-inoculated with a sterile 10 mM MgCl2 solution. The experiments were replicated thrice and inoculated plants were maintained at room temperature with natural sunlight and humidity, which resembled the field conditions. Two months after inoculation, all of the UTAR-BC1 inoculated spots of banana plantlets showed severe necrosis, while the banana leaves showed symptoms of wilted appearance, whereas the control plants remained symptomless. The reisolated pathogen from 90% of the symptomatic pseudostems and leaf blades shares the same morphological and molecular features as UTAR-BC1, thus fulfilling Koch's postulates. Previously, K. variicola has been reported to be a banana pathogen causing rhizome rot in India (Loganathan et al., 2021), plantain soft rot in Haiti (Fulton et al. 2020), and sheath rot and bulb rot in China (Sun et al., 2023; Jiang et al., 2024). To the best of our knowledge, this is the first report of bacterial wilt disease in bananas attributed to K. variicola in Malaysia. This finding will facilitate the surveillance of K. variicola as an emerging pathogen in banana plants in this region, thereby safeguarding the country's food security and promoting socio-economic growth.

Complete genome sequencing of Agrobacterium leguminum strain EL101 isolated from Khaya senegalensis tree bark using nanopore technology.

We report the complete genome sequence of Agrobacterium leguminum strain EL101, isolated from the tree bark of Khaya senegalensis in Kampar, Perak, Malaysia, obtained using Q20+ Nanopore Sequencing chemistry. The assembled genome has a total length of 5,324,685 bp, comprising a circular chromosome, a linear chromid, and two non-Ti circular plasmids.

Investigation of Upper Respiratory Carriage of Bacterial Pathogens among University Students in Kampar, Malaysia.

The carriage of bacterial pathogens in the human upper respiratory tract (URT) is associated with a risk of invasive respiratory tract infections, but the related epidemiological information on this at the population level is scarce in Malaysia. This study aimed to investigate the URT carriage of Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa among 100 university students by nasal and oropharyngeal swabbing. The presence of S. aureus, K. pneumoniae and P. aeruginosa was assessed via swab culture on selective media and PCR on the resulting isolates. For S. pneumoniae, H. influenzae and N. meningitidis, their presence was assessed via multiplex PCR on the total DNA extracts from chocolate agar cultures. The carriage prevalence of H. influenzae, S. aureus, S. pneumoniae, K. pneumoniae, N. meningitidis and P. aeruginosa among the subjects was 36%, 27%, 15%, 11%, 5% and 1%, respectively, by these approaches. Their carriage was significantly higher in males compared to females overall. The S. aureus, K. pneumoniae and P. aeruginosa isolates were also screened by the Kirby-Bauer assay, in which 51.6% of S. aureus were penicillin-resistant. The outcomes from carriage studies are expected to contribute to informing infectious disease control policies and guidelines.

Development of constitutive and IPTG-inducible integron promoter-based expression systems for Escherichia coli and Agrobacterium tumefaciens.

Broad host range (BHR) expression vector is a vital tool in molecular biology research and application. Currently, most of the plasmid vectors used in Agrobacterium spp. are binary vectors that are designed for plant transformation, and very few are designed for expressing transgenes in Agrobacterium spp. Class 1 integrons are common genetic elements that allow for the efficient capture and expression of antibiotic resistance genes, especially in Gram-negative bacteria. One of its compound promoters, PcS + P2, was used in this study and has been reported to be the strongest class 1 integron constitutive promoter; it is referred to as "integron promoter" (P int) henceforth. Herein, we created two versions of isopropyl-d-thiogalactopyranoside (IPTG)-inducible promoters by substituting and/or inserting lacO sequence(s) into P int. These inducible promoters, which possess different degrees of stringency and inducibility, were used to construct two broad host range expression vectors (pWK102 and pWK103) based on the versatile pGREEN system. This allows them to be stably maintained and replicated in both Escherichia coli and Agrobacterium tumefaciens. Functional validation of these vectors was performed by the expression of the reporter gene, superfolder green fluorescent protein (sfGFP), which was cloned downstream of these promoters. Due to the strong induction and tunable expression of a transgene located downstream to the inducible integron promoter, these vectors may be useful for heterologous gene expression in both E. coli and A. tumefaciens, thus facilitating recombinant protein production and genetic studies in Gram-negative bacteria. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03507-0.