Abstract: Five medicinal plants, two agricultural crops grown in different agroclimatic conditions and one weed plant were tested for the presence of endophytic bacteria in their root and stem tissues. The plants tested were Cymbopogon citratus, Azadirachta indica, Phyllanthus emblica, Boerhaavia diffusa, Boerhaavia repens, Pisum sativum, Sorghum bicolor and Parthenium hysterophorus. Sixty different endophytic bacterial isolates belonging to different genera were isolated. Growth promotion ability of these endophytic bacteria was tested on seed germination, seedling vigor and biomass of pearl millet crop. The result indicated that, among sixty isolates tested, ten endophytic bacteria showed significant growth promoting effects on the pearl millet crop plants tested. The same endophytes when coated to seeds of pearl millet, the resulting seedlings showed resistance against downy mildew disease caused by Sclerospora graminicola, an oomycetes pathogen. Apron 35 SD and Benzothiadiazole (BTH) seed treatments were compared with endophytic bacterial treatments. Among the different endophytic bacteria, Pseudomonas fluorescens ISR 34 and Bacillus sp. ISR 37 showed highest protection of 68 and 63%, respectively. Experimental evidences have been provided to show that resistance offered in pearl millet against downy mildew disease is by induced systemic resistance phenomenon.
INTRODUCTION
Endophytic microorganisms are ubiquitous in plants and colonize internal tissues without causing any substantive harm or gaining benefit other than securing residency (Kado, 1992; Bell et al., 1995; McInroy and Kloepper, 1995). They have been found in numerous plant species with most being members of common soil bacterial genera such as Pseudomonas, Bacillus and Azospirillum (Chanway, 1998). Several reports indicated that endophytic bacteria are involved in promotion of plant growth (Chanway, 1998; Hallmann, 2001; Gutierrez-Zamora and Martinez-Romero, 2001; Roncato-Maccari et al., 2003a,b), accumulation of pathogenesis related proteins, deposition of cell wall barriers, (Benhamou et al., 1996), inhibit growth of pathogens by producing antimicrobial compounds like siderophores, (Chen et al., 1995). The capability of colonizing internal host tissues of plant has made endophytic bacteria valuable for agriculture as a tool to improve crop performance especially for those bacteria having commercial features such as plant growth promotion and activation of plant defense mechanisms (Hallmann, 2001). Several studies have indicated that treatment with selected endophytic bacteria could induce resistance against vascular pathogens and also enhance plant growth promotion (Chen et al., 1995; Tuzun and Kloepper, 1995; Hallmann et al., 1997; Benhamou et al., 1998; Manjula et al., 2002).
In the present investigation, pearl millet [Pennisetum glaucum (L.) R.Br.] crop was considered for studies because of its increasing importance as a food and forage crop worldwide. The research efforts are intensified to meet the production constraints originating from biotic and abiotic stresses. One such major biotic constraint is the downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroeter, which gains prime importance due to limited host resistance and is presently managed by metalaxyl fungicides (Singh and Shetty, 1990). The use of agrochemicals, although controls the attack of phytopathogens, but pose a high risk to human health, development of resistance strains, environmental pollution as well as economical feasibility. The ecofriendly approaches such as biological control and host resistance induction have gained much attention in the past decades as a way of reducing the use of chemical products in agriculture. Hence, the efficiency of endophytic bacteria from different plant origin were tested to promote growth and to control downy mildew disease in pearlmillet.
MATERIALS AND METHODS
Isolation of Endophytes
Endophytic bacteria were isolated from stem and root regions of selected plants.
The plant parts were washed thoroughly in running tap water and surface sterilized
with sodium hypochlorite (2%) containing 0.1% Tween 20 for 10 and 60 sec for
stems and roots, respectively. The disinfectant was removed by rinsing five
times each in two washes of sterile distilled water and finally in sterile water
and plant parts were dried on sterile paper towels (Hallmann et al.,
1997; Zinniel et al., 2002) dissected into 1 cm pieces and then pressed
onto nutrient agar as a disinfestations control. Roots and stems were macerated
with sterile mortar and pestle. Tissue extracts were then serially diluted in
12.5 mM potassium phosphate buffer (pH 7.0) and plated in triplicate to recover
any bacterial endophytes present in the plant tissue. For further experiments
different isolates of endophytic bacteria was selected only those samples for
which the disinfestations controls lacked any bacterial growth after incubation
at 28°C for 24-48 h. Sixty isolates were recovered
and reisolated and subjected to gram staining and were used for seed germination
and seedling vigor test. Bacteria isolated were further identified after greenhouse
experiments based on morphology characters such as color, form, elevation, margin,
diameter, surface, opacity, texture, endospore and size and biochemical tests
like catalase test, KOH solubility test and oxidase test (Sneath et al.,
1986) (Table 1). Bacteria were stored at -40°C until
further experiments.
Host and Pathogen
Pearl millet seeds cv. 7042S susceptible to downy mildew were obtained from
All India Co-ordinated Pearl Millet Improvement Project (AICPMIP) and used for
the studies.
| Table 1: | Endophytic bacteria recovered from root and stem regions of different plant species |
Sclerospora graminicola isolated and maintained on pearl millet under greenhouse conditions at the Department of Studies in Applied Botany and Biotechnology, University of Mysore, Mysore, India was used for all experiments.
Growth of Bacteria/Seed Treatment
Bacterial cultures were grown in nutrient broth for 48 h to obtain spore\cells
for use as seed treatment. Bacterial suspensions were centrifuged at 6,000 revolutions
per min (rpm) for 5 min and the pellet obtained was resuspended in sterile distilled
water. The optical density of the suspension was spectrophotometrically and
at 610 nm (Hitachi U-2000, Japan) and adjusted to obtain density of 1x108
cfu mL-1 (Niranjanraj et al., 2003; Umesha et al.,
1998). Seeds of pearl millet were surface sterilized with 2% sodium hypochlorite
(2 min) and washed in sterile distilled water, dried on sterile blotter paper
and soaked in the bacterial suspension (1x108 cfu mL-1)
at 25±2°C for 6 h at 150 rpm in rotary shaker. Seeds treated with
sterile distilled water served as control. Seeds treated with 0.75% BTH (Benzothiadiazole)
and Apron 35SD at the rate of 6 g kg-1 seeds served as positive controls
for greenhouse and field screening.
Effect of Seed Treatment with Endophytic Bacteria on Seed Germination and
Seedling Vigor
Germination test was carried out according to International seed testing
association (ISTA, 2003) and vigor index (VI = Mean Root Length + Mean Shoot
Length x % Germination) was calculated at the end of 7 days (Abdulbaki and Anderson,
1973). Four replicates of 100 seeds were used per treatment. Seeds treated with
endophytic bacteria (1x108 cfu mL-1) were placed
on moist germination paper and incubated at 26±2°C. Seeds treated
with sterile distilled water served as control.
Effect of Seed Treatment with Endophytic Bacteria on Growth Parameters of
Pearl Millet
Seeds treated with endophytic bacterial isolates as well as sterile distilled
water were sown to clay pots containing a sterilized mixture of 5 kg of sand,
soil and manure (1:2:1) and maintained under green house conditions for vegetative
growth parameter studies. Vegetative growth parameters such as height, fresh
weight, dry weight and number of basal tillers per plant were recorded after
30 days of growth. The fresh weights of the plants were determined by weighing
the individual plants immediately after harvesting. Dry weight was estimated
after drying the plants at 65°C in an oven for 12 h. The treated pearl millet
plants were also observed for (a) Total number of productive tillers (b) Number
of days required to 50% flowering (c) height of the plant during flowering (d)
Length and girth of the ear head and (e) 1000 seed weight and in comparison
with untreated plants.
Effect of Seed Treatment with Endophytic Bacteria on Downy Mildew Disease
Incidence under Green House Conditions
For inoculum preparation, leaves of infected pearl millet plants were collected
in the evening hours and washed in running tap water to remove fungal spores
which was then blot dried and placed in a moist chamber for sporulation. Fresh
sporangia were collected the next morning and zoospores released by them were
adjusted to 4x104 zoospores mL-1 and used as inoculum.
Seeds of pearl millet cv. 7042S were treated with endophytic bacterial isolates,
BTH, Apron 35 and sterile distilled water for 6 h and sown into clay pots as
explained above and maintained under green house conditions. Coleoptiles of
two-day old seedlings were inoculated with a suspension with 4x104
zoospores mL-1 of S. graminicola by whorl inoculation method
(Singh and Gopinath, 1985). Each treatment consisted of 25 plants (per treatment)
in four replications.
Demonstration of Induced Resistance by Time Gap Studies in Pearl Millet
Seeds treated with endophytic bacterial isolates, BTH (benzothiadiazole)
and sterile distilled water for 6 h were sown into clay pots as explained above
and maintained under greenhouse conditions.
Coleoptiles of two-day-old seedlings were inoculated with a suspension of 4x104 zoospores mL-1 of S. graminicola as described above. A time gap of 1, 2, 3, 4 and 5 days was maintained between the endophytic bacteria treatment and inoculation with the pathogen S. graminicola. Each treatment consisted of 25 plants in four replicates.
Effect of Seed Treatment with Endophytic Bacteria on Downy Mildew Disease
Incidence under Field Conditions
Field trial was carried out by seed treatment with different endophytic
bacteria and BTH in the pearl millet experimental plot of the Department of
Applied Botany and Biotechnology, University of Mysore, Karnataka, India. Apron
35SD was used for comparing the performance of all treatments along with sterile
distilled water control (Williams et al., 1981). The plot size was 10x5
m and recommended normal agronomic practices were followed during the trial.
Plant-to-plant distance of 15 cm and row-to-row distance of 45 cm was maintained.
The experiment was designed as a random block design with four replicates.
Disease Assessment
After seven days, the plants were observed daily for expression of downy
mildew disease. Plants were rated diseased when they showed typical symptoms
of downy mildew, i.e., sporulation, stunting, chlorosis and green ear (Shetty
et al., 1995). The disease incidence was recorded at 30 and 60 days after
sowing.
Data Analysis
All the experimental results were suscitated to Drican’s Multiple Range
Test (DMRT). The means were compared for significance using DMRT (p = 0.05).
RESULTS
Isolation of Endophytic Bacteria
Five medicinal plants, two agricultural crops with two different varieties
and one weed plant were tested for the presence of endophytic bacteria. The
medicinal plants used were C. citratus, A. indica,
P.emblica, B. diffusa and B. repens, two cultivars
of P. sativum and S. bicolor and P. hysterophorus.
C. citratus is a member of Graminae used for scented oil. A.
indica is used for wide range of products such as pesticides and human health
care. P. emblica is a rich source of antioxidants. B.
diffusa and B. repens are used in herbal medicine to correct liver
disorders. P. sativum and S. bicolor are important
legume and cereal crop, respectively and harbor wide range of bacteria in root
and stems. P. hysterophorus is a weed with plenty of allelopathic
chemicals secreted in the rhizosphere.
Sixty endophytic bacteria were isolated from all the plant species tested. From each plant species bacteria isolated were different and they were identified based on gram staining and biochemical tests. Out of sixty isolates, ten isolates showed promising effects on pearl millet growth and were used for further experiments. C. citratus isolate was identified as Bacillus sp. ISR 38, A. indica as P. fluorescens ISR 36, P. emblica as Bacillus sp. ISR 42, B. diffusa as Bacillus sp. ISR 40 and B. repens as P. fluorescens ISR 34, was observed. In P. sativum revealed Bacillus sp. ISR 39 and P. fluorescens ISR 33. S. bicolor revealed P. fluorescens ISR 35 and Bacillus sp. ISR 41, respectively. Bacillus sp. ISR 37 was isolated from P. hysterophorus.
Effect of Seed Treatment with Endophytic Bacteria on Seed Germination and
Seedling Vigor
In general, all the endophytic bacterial isolates significantly enhanced
the seed germination and seedling vigor of pearl millet. However, the degree
of enhancement varied between the bacterial treatment.
| Fig. 1: | Effect of seed treatment with promising endophytic bacteria on seed germination and seedling vigor of pearl millet |
The highest germination percentage and vigor index was recorded for the Pseudomonas isolates. P. fluorescens ISR 33 and P. fluorescens ISR 36 achieved 96% germination followed by Bacillus sp. ISR 37 and P. fluorescens ISR 35 with 94% germination, which was significantly higher when compared with the control (87.5%). The lowest percent germination of 87 was observed in Bacillus sp. ISR 39 ( Fig. 1). The similar trend was also noticed for the seedling vigor in which all the bacterial isolates enhanced the vigor index to varied degrees. The highest seedling vigor 1507 was recorded for the treatment with P. fluorescens ISR36, followed by P. fluorescens ISR33, Bacillus sp. ISR37, ISR35, ISR 38, ISR 42, ISR 40, ISR34, ISR41 and ISR39 ranged from 1482-1292 while control showed 1263.
Effect of Seed Treatment with Endophytic Bacteria on Growth Parameters of
Pearl Millet
All the tested endophytic bacterial isolates were significant in promoting
the vegetative growth parameters of pearl millet plants. The vegetative growth
parameters such as height of the plant, fresh weight, dry weight and number
of basal tillers were significantly increased over control due to seed treatment
with endophytic bacteria. Maximum height of 42 cm was recorded in plants treated
with P. fluorescens ISR 36 compared to 31 cm in control. The Bacillus
isolates enhanced the plant height significantly, which ranged from 35-40
cm. P. fluorescens ISR36 and Bacillus sp. ISR 37 recorded the
maximum fresh weight of 17 g and 16.8 g, respectively while control plants showed
fresh weight of 10 g. Similar trend in improvement was also noticed for dry
weight. Further, increase in the number of tillers per plant was evident in
all endophytic bacterial treatments. P. fluorescens ISR 36 and Bacillus
sp. ISR 37 recorded maximum of 4.5 tillers in comparison to 2.0 tillers
in the control (Table 2).
Similar to the vegetative growth parameters offered by the endophytic bacterial
isolates were also enhancing the reproductive growth parameters such as 50%
flowering, length and girth of ear heads and 1000 seed weight, plant height,
tillering of pearl millet plants (Table 3). The maximum improvement
of all reproductive traits was noticed in plants raised from seeds treatment
with P. fluorescens ISR 36 subsequently, ISR 35, ISR 37, ISR 38 isolates.
All the endophytic bacterial isolates reduced the 3-4 days following by compared
to untreated control plants.
| Table 2: | Effect of seed treatment with promising endophytic bacteria on vegetative growth parameters of pearl millet plants cv.7042S |
| Note: Results were taken 30 days after sowing and are based on the four replicates with 100 plants per treatment; Means followed by the same letter are not significantly different according to DMRT (p≤0.05); SDW-sterile distilled water | |
| Table 3: | Effect of seed treatment with promising endophytic bacteria on reproductive growth parameters of pearl millet cv.7042S |
| Results were taken 60 days after sowing and based on two replicates with 50 plants per treatment; Means followed by the same letter are not significantly different according to DMRT (p≤0.05); SDW-Sterile distilled water | |
| Fig. 2: | Effect of seed treatment with endophytic bacteria on downy mildew protection under green house conditions (DMDI - Downy mildew disease incidence, DMDP- Downy mildew disease protection) |
Effect of Seed Treatment with Endophytic Bacteria on Pearl Millet Downy
Mildew Incidence Under Greenhouse Conditions
All the endophytic bacterial isolates were efficient in reducing the downy
mildew incidence compare to control under greenhouse conditions. However, the
degree of disease reduction varied with the isolates tested ranging from 15
to 53%. Maximum protection of 53% was offered by P. fluorescens ISR36
followed by 51, 49.5, 49, 43% protection by Bacillus sp. ISR40, P.
fluorescens ISR33, Bacillus sp. ISR37, Bacillus sp. ISR 38,
respectively. However, none of the treatments were on par to BTH (68%) or Apron
(92%) treatments in offering protection against downy mildew disease ( Fig.
2).
Demonstration of Induced Resistance by Time Gap Studies in Pearl Millet
In order to test the nature of resistance offered by endophytic bacterial
isolates, induction studies were conducted by treating seeds with the bacteria
and inoculating with the pathogen at different time intervals.
| Fig. 3: | Effect of seed treatment with endophytic bacterial isolates due to inoculation with Sclerospora graminicola, DM-Downy mildew |
| Fig. 4: | Effect of seed treatment with endophytic bacteria on downy mildew disease protection under field condition (DMDI-Downy mildew disease incidence, DMDP-Downy mildew disease protection) |
After seed treatment with endophytic bacteria, protection of 14-68% was observed, depending on the time interval between bacteria treatment and inoculation with pathogen. Maximum protection was achieved by the isolate P. fluorescens ISR 34, which recorded 68% protection on 4th and 5th day of time gap between seed treatment and pathogen inoculation ( Fig. 3). On the first day this isolate recorded 44%, which was raised to 66 and 65% on the 2nd and 3rd day, respectively followed by Bacillus sp. ISR 40, ISR 37, P. fluorescens ISR 33, P. fluorescens ISR 36 showed 63, 58, 54%, respectively. The lowest protection was recorded by Bacillus sp. ISR 41, which recorded 14% protection. BTH, which was used as a positive control offered the highest protection of 83.5% in comparison to other treatments and the control.
Effect of Seed Treatment with Endophytic Bacteria on Pearl Millet Downy
Mildew Incidence under Field Conditions
Under field conditions, all the endophytic bacterial isolates showed reduced
downy mildew incidence, but the degree of protection varied between isolates.
The protection offered due to seed treatment ranged from 18 to 55%. Maximum
protection by P. fluorescens ISR36 followed by Bacillus sp. ISR37,
ISR 40, ISR 38 with 52.5, 51 and 46.5%, respectively. The lowest protection
was recorded for Bacillus sp. ISR41 with 18%. However, BTH or Apron provided
even better protection, with 68 and 94%, respectively ( Fig. 4).
DISCUSSION
Medicinal plants, agriculturally important crops and weed plants harbor different types of microorganisms on the surface as epiphytes or endophytes. Epiphytic microorganisms are associated on the surface of the plants may have beneficial or harmful effects to the plants. However, the endophytic microbes show beneficial effects to the harboring plants. The secondary metabolites in medicinal plants may be elicited due to the presence of endophytes.
Endophytic bacteria isolated in the present study have shown different beneficial effects to the pearl millet plants, which is grown as important cereal crop of the semi-arid tropics. Most of the endophytic bacteria isolated in the study belonged to Bacillus and Pseudomonas species. Results of the present study evidenced the fact that seed treatment with endophytic bacteria enhanced seed germination and seedling vigor of pearl millet. The germination percentage of 96% and seedling vigor of 1507 was offered by P. fluorescens ISR 36. There are no reports of the effect of endophytic bacteria on seed germination and seedling vigor. This study also showed the endophytic bacteria isolated from A. indica showed considerable influence on seed germination and seedling vigor enhancement. Similarly, seedling vigor enhancement has been noticed with seed treatment of pearl millet by different endophytic bacteria isolated from medicinal, agricultural and weed plants.
Seeds treated with endophytic bacteria showed increased fresh weight and dry weight of the plant, more number of tillers, early flowering and 1000 seed weight. Maximum plant biomass enhancement was offered by P. fluorescens ISR 36. There are a few reports on the increased plant biomass of different crop species such as oilseed rape, tomato, maize, sorghum, wheat and rice when endophytic bacteria used as seed and seedling treatments (Nejad and Johnson, 2000; Gutierrez-Zamora and Martinez-Romero, 2001; Roncato-Maccari et al., 2003a,b). This phenomenon has been attributed to microbial processes leading to nutrient solubilization by production of phosphorous and siderophores and plant growth hormones such as auxins, cytokinins, gibberlins, abscisic acid (Sturtz et al., 1997).
Significant reduction of downy mildew disease of 53 and 55% were recorded under greenhouse and field conditions, respectively due to seed treatment with endophytic bacteria. The protection offered by the endophytes due to seed treatment was tested for induction of resistance in the plant by time gap studies between seed treatment and pathogen inoculation. In several case studies, an endophytic bacteria Bacillus pumilus SE34against root rot causing fungus Fusarium oxysporum f. sp. pisi in Pisum sativum and increased resistance against Fusarium oxysporum f. sp. lycopersici in tomato has been recorded(Benhamou et al., 1996; M’Piga et al., 1997). Further, in rice against Rhizoctonia solani (Krishnamurthy and Gnanamanickam, 1997), cotton against Verticillium dahliae (Xia et al., 1996) and wilt diseases of oil seed rape and tomato have shown the pretreatment of endophytic bacteria has resulted in increased host defense responses (Nejad and Johnson, 2000). Although, the exact mechanism by which seed treatment with endophytic bacteria reduces disease incidence are not fully understood, it has been reported that endophytic bacteria are known to control plant pathogens by induction of resistance leading to the production of phytoalexins, accumulation of pathogenesis related proteins, deposition of structural barriers in the cell wall of the host plant and by production of antimicrobial compounds (Manjula et al., 2002).
Plant Growth Promoting Rhizobacteria (PGPR) have been well documented to improve the growth promotion and host resistance against the downy mildew pathogen in pearl millet (Niranjanraj et al., 2003). Endophytic bacteria are often closely related to PGPR/Plant Health Promoting Rhizobacteria (PHPR) and these have lately found to colonize the root internally also indicated modes of action described for PGPR/ PHPR also apply for endophytic bacteria. These endophytic bacteria induced resistance can last longer than PGPR, since they establish a much closer relationship with the host and on the other hand, PGPR may be inhibited by competition with other microorganisms on the root surface (Hallmann, 2001). The penetration of endophytic bacteria into the seeds or sprouting seedlings leading to defense elicitation is most likely the possible explanation.
Seed treatment is the only feasible technology in pearl millet due to economic constraints for crop production. Alternative is the exploitation of biotic and abiotic inducers, which have plant growth promoting effects and also induction of resistance against pests and diseases. The use of endophytes may be preferable to reduce the use of chemical fertilizers and pesticides because of cost and contribution to sustainable agricultural systems.
ACKNOWLEDGMENTS
This study has been carried out in the project on ‘Systemic Acquired Resistance’ funded by Danish International Development Agency under the Enhancement of Research Capacity Programme (DANIDA ENRECA). We are grateful to Dr. Eigil de Neergaard, the Principal Responsible Party of the DANIDA ENRECA project for his cooperation during the study. The facilities provided by Indian Council of Agricultural Research (ICAR), Government of India through All India Coordinated Pearl Millet Improvement Programme (AICPMIP) is also gratefully acknowledged.