Plant growth promotion and plant protection through plant microbe interaction has been given considerable interest by researchers and phylloplane microorganisms were explored for the last two decades. Recent studies showed that plants constitute diverse niches of endophytic microorganisms inhabiting in tissues without damaging their hosts. The relatively stable environment inside the plant tissues bioactive than the rhizosphere makes endophytes more associated microorganisms (He
In sugarcane endophytic bacteria inoculation is reported to increase biological nitrogen fixation, promotion of root development, increased biomass and productivity (Oliveira
In mulberry, plethora of chemical are recommended to combat various diseases. However increased environmental awareness, cost of chemicals and highly sensitive nature of silkworm to plant protection chemicals that a minute chemical residue sufficient to result huge crop loss, make farmers reluctant to use chemicals. The alternative methods are therefore very essential to contain the situation. Considering the broad spectrum beneficial effect of endophytic bacteria, the present study attempt to isolate and evaluate few mulberry endophytic bacteria and evaluate their potential to control two fungal pathogens associated with root rot disease of mulberry.
The nutrient agar medium with pH 7.0 was used for isolation of endophytic bacteria. The medium and plates were sterilized at 121℃ for 20 min and cooled to 50℃. The sterile plates were exposed to UV light for 15 min. About 15 mL media were poured in 9 cm dia. Petri plates, the plates were then exposed to UV light for 15 min to ensure the sterility. The isolation of endophytes was done according to the procedure by Bacon
The outer layer of the cortex of the sample is peeled using sterilized sharp knife. Small pieces (0.2 cm) were taken from the inner cortical region of the cuttings these pieces were aseptically placed on sterile nutrient agar. The plates were incubated in BOD incubator at 27±2℃ for 72 h in inverted position. To ensure the sterility of the samples, the final wash collected were streaked on nutrient agar plates and incubated.
The individual colonies grown on nutrient agar were sub-cultured in nutrient media and again transferred to the nutrient agar slants. These were considered as mother culture and used to study cultural and morphological characters. Morphological cha racteristics such as colony colour, size, margin, consistency and texture of all the isolates were observed. From the total isolates, based on colony morphology, limited number of representative isolates was selected for further investigation. All the selected isolates were sub-cultured in nutrient agar slants and preserved in 4℃ for further investigation. Also, preliminary phenotypic as well as microscopic characterization of bacteria such as Gram reaction, endospore staining, motility, catalase and oxidase activity of all the isolates were performed by adopting standard procedure (Tiwari
The pure cultures of endophytes were inoculated on nutrient agar plates and left for incubation in BOD incubator at 27 ±2℃ for 72 h, The bacterial suspension was prepared from these pure cultured by adding 5 mL of sterile distilled water to plates and gently mixed using a camel hair brush. The suspensions were taken in test tubes separately for each isolates and added sterile distilled water. The optical density (OD) of the suspension was checked and adjusted with blank to make OD 600 under spectrophotometer so as to get approximately 8×108 cells/mL.
For bio priming of mulberry seeds, good quality seeds of mulberry var. S799 OP variety were collected from Mulberry Breeding and Genetics Department CSR&TI, Mysore. About 300 seeds were taken in 50 mL test tube containing 10 mL bacterial suspension. These seeds in soaked condition was left for 12 h. The seeds soaked similar way in sterile distilled water served as blank. Thereafter the soaked seeds were spread on Petri plates (15 mm dia.) lined with moistened filter paper and kept for germination. To filter paper was moistened with distilled water to avoid drying using a dropper. Germination was considered when the seeds developed at least 2 mm long radical. The germination percentage was calculated based on the total number of seeds germinated on after seven days. The weight and length of root, shoot and total length of the seedling were recorded from each treatment and control.
All bacterial isolates were screened for their ability to inhibit fungal growth on PDA plates using dual culture technique (Yoshida
The percentage data were subjected for arcsine transformation before analysis of variance (ANOVA) and mean values were compared for significant difference.
Among 10 endophytic bacteria isolated, except two isolates, all were colourless and two isolates were yellow. The colony size of eight isolates was small and two were large. The margin of five isolates was irregular while margin of five isolates were circular. The consistency of three isolates was creamy and for six isolates it was slimy and for one isolate was mucoid. Texture of seven isolates was convex and of the rest was flat (Table 1).
Cultural characterization of endophytic bacteria
The gram staining showed that the eight isolates as Gram positive and the rest Gram negative. Regarding the shape, five isolates were cocci and others were bacilli (rod shaped). Four isolates were showed mobility and all were catalase positive. However three isolates En-3, En-5 and En-7 were oxidase positive. Spore staining was positive only in isolates En-4 and En-10 (Table 2).
Microscopic characterization of endophytes
Except three isolates, treatment with all other isolates increased seed germination compared with untreated control. A higher germination (>96%) was observed due to biopriming with bacterial isolates, En-7 (98.45%), En-10 (97.26%), En-5 (97.34%), and En-2 (96.14%). The germination in case of three isolates was less than that of control (Fig, 1).
There was significant difference in root length and seedling length. However, no significant difference observed in shoot length and seedling weight. The seedling weight was found higher (0.013 g) in case of seeds treated with bacterial isolates En-5 and En-7, followed by En-2 and En-10 (0.12). The least weight was observed in case of treatment with En-9 (0.007 g). Similarly, shoot length was more in seeds treated with isolate En-7 (24.50 mm) followed by En-8 (23.9 mm) treated lots and was least in case of isolates En-3 and En-9 (21.18 mm) which were less than that of control (22.17 mm).
The antagonistic effect of the bacterial isolates was tested against
Influence of endophytic bacteria on seedling growth of mulberry
Inhibition of mycelia growth of R. bataticola by endophytic bacteria
The bacterial isolates and interaction between bacterial isolates and days after inoculation significantly (
Inhibition of mycelia growth of F. oxysporum by endophytic bacteria
Bacterial endophytes are present in most of the plants (McInroy and Kloepper, 1995; Sturz, 1995). The beneficial effects of bacterial endophytes on their host plant appears to occur through similar mechanisms as described for rhizosphere-associated bacteria. These mechanisms have been reviewed in great detail by Kloepper
As an endophyte, motility is an advantage for moving inside the plant system. The present study showed that 40% of the bacteria were highly motile. Interestingly, the proportion of Gram positive isolates was found predominant Endophytic bacteria residing within plant tissues have been reported to be promoting the plant growth directly or indirectly. The growth stimulation due to endophyte association can be as a consequence of nitrogen fixation (Hurek
Endophytic bacteria are able to lessen or prevent the deleterious effects of certain pathogenic organisms. Endophytes from potato plants showed antagonistic activity against fungi (Berg
The study reveals that there are many endophytic bacteria in mulberry which are beneficial to the plant by helping in growth promotion and also antagonistic to the pathogens associated with root rot disease of mulberry. These endophytic bacterial isolates could be explored for improving mulberry growth and protection from soil borne diseases.