A local Bacillus spp.
Authors: Mohamed Shereen A.H. , El-Sayed Ghada M. , Elkelany Usama S. , Youssef Mahmoud M.A. , El-Nagdi Wafaa M.A. , Soliman Gaziea M. 
Published: August 2024

Background
Increasing chemical nematicide and fertilizer application has disturbed the natural balance and increased human health risks, which in turn compelled and increased the urgency to discover novel and more safe and effective biological tools to control root-knot nematode (Meloidogyne incognita) distributed throughout Egypt.

Objective
Isolation and screening of rhizobacteria from soil samples was the first step. The second step was the evaluation of the isolated rhizobacteria capability as nematicides under laboratory and screenhouse conditions. The third was to employ protoplast fusion as genetic technique to improve the nematicidal effect of rhizobacterial bacteria strains and comparison between parent and fusant protein profiles to analyze the genetic recombination among them.

Materials and methods
Serial dilution method was used to isolate rhizobacteria on Tryptic Soy Agar plates. The evaluation test against M. incognita J2 of bacterial isolates was achieved under laboratory conditions and nematode parameters under screenhouse. The most potent rhizobacteria were exposed to molecular identification using 16s rDNA sequencing. Bacillus thuringiensis subsp tenebrionis (Bt) was evaluated for the first time as a bioagent that acted as a nematicide. Protoplast fusion was employed between the most potent bacterial strain, Bacillus cereus (Bc) and B. thuringiensis subsp tenebrionis (Bt). Selection of bacterial fusants was achieved on complete media containing selectable antibiotics marker. Parental and fusant strains were evaluated against J2 under laboratory and screenhouse conditions and their ability in plant-growth promotion by assessment of plant parameters. SDS-PAGE analysis was used to analyze protein profiles.

Results and conclusion
Out of 15 bacterial isolates, NRC12 recorded the highest percentage mortality 88.3% after 24 h compared with control. NRC12 was identified as B. cereus NRC12 (Bc) and deposited in Genbank under accession number MW548408. Protoplast fusion between B. cereus (Bc) and B. thuringiensis (Bt) resulted in formation of 10 stable bacterial fusants that gave higher nematicidal effect compared with parental strains. Bacterial fusant, F7, achieved the highest mortality and reduction in J2 in soil by 98.3 and 87.19% under laboratory and screenhouse conditions, respectively. The highest reduction in the number of galls and egg masses on root system was also recorded by F7 with 77.18 and 72.35%, respectively, compared with its parents. Also, F7 registered the highest significant (P≤0.05) increment in length, fresh weight, and dry weights in the shoot system of eggplant in pot experiments. The bacterial fusant, F7, exhibited more ability to fix atmospheric nitrogen compared with bacterial parents. Protein profile of F7 proved that this fusant was the only one that acquired equal protein bands from each parent. The highest frequency of genetic recombination might occur in F7 and subsequently, induced effective nematicidal effect more than its parents.