Isolation and determination of bacterial microbiota of Varroa destructor and isolation of Lysinibacillus sp. from it

Background The importance of bees for environmental health is known. Within the scope of this importance, it is of great importance to protect the health of bees and to prevent colony extinction. In this context, it is very important to develop effective methods in combating microorganisms, parasitoids, mites and organisms that cause disease or harm in bees. Both use different methods in terms of bee health. Result In this study, the possibility of the bacteria isolated from Varroa destructor mite being bioinsecticide was investigated. Accordingly, six bacteria were isolated from the mite. Isolated bacteria were analyzed according to biochemical tests, molecular analysis, optimum growth pH and phylogenetic tree drawn as Pantoea dispersa (GV1), Lysinibacillus macroides (GV3), Bacillus mycoides (GV4), Lysinibacillus fusiformis (GV5), Pseodomonas lutea (GV5), Lysinibacillus varians (GV7). Lysinibacillus sp. The entomopathogenic feature of Lysinibacillus sp. ranked it as the most important species. When the insecticidal properties of bacteria were examined, they were determined as 53, 90, 62, 95, 74 and 83% for GV1, GV3, GV4, GV5, GV6 and GV7, respectively. Conclusion Based on these results and literature review, Lysinibacillus sp. species had a high potential to be used as bioinsecticide against V. destructor mite.

Diversity of midgut bacteria in larvae and females of Aedes aegypti and Aedes albopictus from Gampaha District, Sri Lanka

Background The midgut microbiota of mosquitoes maintain basal immune activity and immune priming. In recent years, scientists have focused on the use of microbial communities for vector control interventions. In the present study, the midgut bacteria of larvae and adults of Aedes aegypti and Ae. albopictus were assessed using both field-collected and laboratory-reared mosquitoes from Sri Lanka. Methods Adults and larvae of Ae. aegypti and Ae. albopictus were collected from three selected areas in Gampaha Medical Officer of Health area, Gampaha District, Western Province, Sri Lanka. Bacterial colonies isolated from mosquito midgut dissections were identified by PCR amplification and sequencing of partial 16S rRNA gene fragments. Results Adults and larvae of Ae. aegypti and Ae. albopictus harbored 25 bacterial species. Bacillus endophyticus and Pantoea dispersa were found more frequently in field-collected Ae. aegypti and Ae. albopictus adults, respectively. The midgut bacteria of Ae. aegypti and Ae. albopictus adults (X2 = 556.167, df = 72, P < 0.001) and larvae (X2 = 633.11, df = 66, P < 0.001) were significantly different. There was a significant difference among the bacterial communities between field-collected adults (X2 = 48.974, df = 10, P < 0.001) and larvae (X2 = 84.981, df = 10, P < 0.001). Lysinibacillus sphaericus was a common species in adults and larvae of laboratory-reared Ae. aegypti. Only P. dispersa occurred in the field-collected adults of Ae. aegypti and Ae. albopictus. Species belonging to genera Terribacillus, Lysinibacillus, Agromyces and Kocuria were recorded from Aedes mosquitoes, in accordance with previously reported results. Conclusions This study generated a comprehensive database on the culturable bacterial community found in the midgut of field-collected (Ae. aegypti and Ae. albopictus) and laboratory-reared (Ae. aegypti) mosquito larvae and adults from Sri Lanka. Data confirm that the midgut bacterial diversity in the studied mosquitoes varies according to species, developmental stage and strain (field vs laboratory). Graphical abstract

First Report of Bud Soft Rot on Agave angustifolia Caused by Pantoea dispersa in Mexico

The agave (Agave spp.) is an important crop in México, with 120,897 ha grown mainly for alcoholic beverage production (SIAP, 2019). In September 2020, in the municipality of Huitzuco de los Figueroa (18.328692 N; 99.3998 W), Guerrero State, México, a serious disease was observed affecting Agave angustifolia. Disease incidence was 8% of 150 plants sampled over an approximate area of 2.5 ha. Initial symptoms of soft rot of the bud developed and produced an abundant exudate which appeared from the apical part to the base of the plant. In severe infections, the plants showed total maceration of the bud, and consequently death of the plants was observed. Symptomatic plant tissue was superficially disinfected with 1% NaOCl for 30 s, and rinsed in sterile water three times. The disinfected tissues were macerated and with a loop spread in Nutrient Agar. The plates were incubated at 28 ° C for 2 days. Yellowish bacterial colonies were isolated, and eight colonies were selected for characterization. The bacterial strains were gram negative and rod-shaped, negative for fluorescent pigment tests and Kovacs' oxidase. Two isolates designated AGA1 and AGA2 were identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F / 1492R (Lane 1991), and partial fusA, rpoB, and gyrB genes (Delétoile et al. 2009). Sequences were deposited in GenBank, with the accession numbers for 16S rRNA, AGA1 as MW548406 and AGA2 as MW548407; for specific genes fusA (AGA1 = MW558445, AGA2 = MW558446), rpoB (AGA1 = MW558447, AGA2 = MW558448) and gyrB (AGA1 = MW558449, AGA2 = MW558450), and they were compared with the sequences available in GenBank using BLASTn. 16S rRNA gene sequences for AGA1 and AGA2 aligned with Pantoea dispersa (MT921704.1, 99.9% identity). Housekeeping genes also aligned 99 to 100% to P. dispersa (fusA = 100%, CP045216.1; rpoB = 99.8% MH015167.1 and gyrB = 99%, MK928270.1). Phylogenetic analysis of concatenated genes showed that strains AGA1 and AGA2 cluster with P. dispersa. To confirm pathogenicity, eight plants of six-month-old A. angustifolia were inoculated with strain AGA1 using sterile toothpicks dipped in 108 CFU/ml bacterial suspension. The toothpicks were inserted in the middle part of the bud. Four plants were inoculated with sterile water as control. The plants were covered with plastic bags and housed in a greenhouse (average temperature and relative humidity of 25 ° C and 85%, respectively). Pathogenicity tests were repeated two times. After seven days, all inoculated plants developed symptoms similar to those observed in the field. Control plants did not show symptoms. From the plants that showed symptoms, the pathogen was reisolated again and was identified by morphological and molecular characterization, following the method previously described, fulfilling Koch's postulates. In México, Erwinia cacticida and Pantoea ananatis has been previously reported on A. tequilana that as causing soft rot and red leaf ring, respectively (Jimenez-Hidalgo et al. 2004; Fucikovsky and Aranda 2006). To our knowledge, this is the first report of P. dispersa causing bud soft rot on A. angustifolia in México. More studies monitoring and control strategies of bud soft rot on A. angustifolia are required.

Diazotrophic Bacteria Pantoea dispersa and Enterobacter asburiae Promote Sugarcane Growth by Inducing Nitrogen Uptake and Defense-Related Gene Expression

Sugarcane is a major crop in tropical and subtropical regions of the world. In China, the application of large amounts of nitrogen (N) fertilizer to boost sugarcane yield is commonplace, but it causes substantial environmental damages, particularly soil, and water pollution. Certain rhizosphere microbes are known to be beneficial for sugarcane production, but much of the sugarcane rhizosphere microflora remains unknown. We have isolated several sugarcane rhizosphere bacteria, and 27 of them were examined for N-fixation, plant growth promotion, and antifungal activity. 16S rRNA gene sequencing was used to identify these strains. Among the isolates, several strains were found to have a relatively high activity of nitrogenase and ACC deaminase, the enzyme that reduces ethylene production in plants. These strains were found to possess nifH and acdS genes associated with N-fixation and ethylene production, respectively. Two of these strains, Pantoea dispersa-AA7 and Enterobacter asburiae-BY4 showed maximum plant growth promotion (PGP) and nitrogenase activity, and thus they were selected for detailed analysis. The results show that they colonize different sugarcane tissues, use various growth substrates (carbon and nitrogen), and tolerate various stress conditions (pH and osmotic stress). The positive effect of AA7 and BY4 strains on nifH and stress-related gene (SuCAT, SuSOD, SuPAL, SuCHI, and SuGLU) expression and the induction of defense-related processes in two sugarcane varieties, GT11 and GXB9, showed their potential for stress amelioration and PGP. Both bacterial strains increased several sugarcane physiological parameters. i.e., plant height, shoot weight, root weight, leaf area, chlorophyll content, and photosynthesis, in plants grown under greenhouse conditions. The ability of rhizobacteria on N-fixing in sugarcane was also confirmed by a 15N isotope-dilution study, and the estimate indicates a contribution of 21–35% of plant nitrogen by rhizobacterial biological N fixation (BNF). This is the first report of sugarcane growth promotion by N-fixing rhizobacteria P. dispersa and E. asburiae strains. Both strains could be used as biofertilizer for sugarcane to minimize nitrogen fertilizer use and better disease management.