APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1987, p. 2056-20590099-2240/87/092056-04$02.00/0Copyright C) 1987, American Society for Microbiology

Evaluation of Pseudomonas fluorescens for Suppressionof Sheath Rot Disease and for Enhancement of

Grain Yields in Rice (Oryza sativa L.)N. SAKTHIVEL AND S. S. GNANAMANICKAM*

Centre ofAdvanced Study in Botany, University of Madras, Guindy, Madras 600 025, India

Received 10 March 1987/Accepted 2 June 1987

Pseudomonasfluorescens strains antagonistic to Sarocladium oryzae, the sheath rot (Sh-R) pathogen of rice(Oryza sativa L.), were evaluated in greenhouse and field tests for suppression of Sh-R severity andenhancement of grain yields of rice. Imprints of rice seedlings and a direct-observation technique of stainingroots with fluorochromes confirmed the association of P. fluorescens with roots and the ability of the strain tomove along shoot tips. In greenhouse tests, P. fluorescens-treated rice plants (cv. IR 20) showed a 54%reduction in the length of Sh-R lesions. In three field tests, treatment with P. fluorescens reduced the severityof Sh-R by 20 to 42% in five rice cultivars. Bacterization of rice cultivars with P. fluorescens enhanced plantheight, number of tillers, and grain yields from 3 to 160%.

In recent years, the heterotrophic rhizobacteria Pseudo-monas fluorescens and Pseudomonas putida have beensuccessfully used as seed inoculants for biological control ofseveral plant pathogens (2, 3, 5-7, 10, 17). These bacteriahave also been used successfully to suppress rice pathogensunder flood conditions [11, 12; N. Sakthivel and S. S.Gnanamanickam, Int. Rice Res. Newsl. 11(3):17-18, 1986].Fluorescent pseudomonads of the plant-growth-promotingrhizobacterial type can significantly increase plant growthand yield under gnotobiotic conditions, as well as in the field(9, 10). The ability of these bacteria to reduce disease andpromote plant growth is believed to be due in part toantibiosis in the rhizosphere and subsequent displacement ofroot-colonizing microflora (9). We have isolated and charac-terized several native P. fluorescens strains which showantagonism toward fungal and bacterial plant pathogens (14).We previously reported evidence which suggested that thesenative strains control the root and stem rot plant pathogens,such as Sclerotium rolfsii, in peanut (5). In the present study,we present results of our evaluation of P. fluorescens strainsuseful for suppressing sheath rot (Sh-R) of rice (Oryza sativaL.), which has become a major constraint to cultivation ofnitrogen-responsive, high-yielding rice cultivars.

MATERIALS AND METHODSBacterial strains. The P. fluorescens strains used in this

study are listed in Table 1. They were isolated from leaf androot samples of rice and of other crops, e.g., black gram,

citrus, cotton, and cowpea, on King's medium B (KB) (8).Fluorescent bacteria were further characterized throughmicrobiological and biochemical tests listed in Bergey'sManual (4).

Fungal isolate. Sarocladium oryzae was isolated fromnaturally infected rice plants with severe Sh-R symptoms.Pure cultures were maintained on potato glucose agar.

Assay for in vitro antibiosis. In vitro tests for antagonism ofP. fluorescens to S. oryzae were made by using plate assays

(13). Bacterial plugs of agar which contained the fluorescent

* Corresponding author.

green pigment (siderophore) were removed from a 48-h P.fluorescens culture. The plugs were transferred to the centerof KB agar plates which had been spray inoculated previ-ously with a spore suspension (106 conidia ml-') of S.oryzae. After the plates were incubated at 23°C for 7 days,inhibition of S. oryzae growth was observed around thebacterial plugs. The P. fluorescens strain that induced thelargest inhibition zones was selected and used in greenhouseand field tests.Root colonization assays. The ability of a P. fluorescens

strain isolated from citrus to associate with rice roots wastested by planting bacterized rice seeds (cv. IR 20) in sterilesand. Seedlings (1 to 7 days old) were lifted from pots, andsoil particles were removed with a soft sterile brush. Theentire seedling was imprinted on KB agar containing strep-tomycin (100 ,ug ml-') for 30 s. Fluorescent bacteria wereobserved to be associated with roots and shoot tips after 48h. By using prints of older seedlings on KB agar, themovement of bacteria from roots to upper parts was exam-ined.To assess root colonization by P. fluorescens, the flu-

orochrome staining technique developed by van Vuurde andElenbass (19) and van Vuurde and Schippers (20) was used.

TABLE 1. Growth inhibition of S. oryzae byP. fluorescens strains

Diam ofStrain (source) inhibition

(cm)a

P. fluorescens (citrus leaf) ............... .......... 2.5P. fluorescens (black gram root)................... 2.3P. fluorescens (cotton root)....... ............ 2.2P. fluorescens (cowpea root) ........ ........... 2.1P. fluorescens 1 (rice rhizosphere)................... 0.85P. fluorescens 2 (rice root) ....... ............ 1.6P. fluorescens 3 (rice rhizosphere)................... 1.5P. fluorescens 4 (rice rhizosphere)................... 0P. fluorescens s (rice rhizosphere)................... 0P. fluorescens 6 (rice rhizosphere)................... 0

" Average of three replications.

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EVALUATION OF P. FLUORESCENS 2057

FIG. 1. Assay for in vitro antibiosis. Plate on right shows growth inhibition of S. oryzae induced by the P. fluorescens strain isolated fromcitrus leaf. Plate on left is the control.

The seminal root segments (20 mm) of rice seedlings (1 weekold) were washed in sterile distilled water and successivelystained with coriphosphine (1:2,000, 15 min), Congo red(1:10,000, 10 min), and acridine orange (1:10,000, 2 min). Toprevent microbial activity, the segments were stored at 4°Cin a solution of cycloheximide (50 ,ug ml-') for 10 min. Thestained segments were washed in sterile distilled water for 10min and mounted on glass slides for observation by fluores-cence microscopy (blue incident light, filter K460).

Bacterization of rice seed. An efficient P. fluorescensstrain, isolated from citrus, which was resistant to strepto-mycin (100 ,ug ml-') was used in these experiments. Cells ofthis strain were grown for 48 h on KB agar, suspended in 1%(wt/vol) carboxymethyl cellulose solution, and adjusted to108 CFU ml-.

Bacterization was done as follows. Rice seeds were mixedwith the P. fluorescens suspension and coated with pow-dered vermiculite (17). Control seeds were similarly treated,but with a coating mixture which did not have bacteria. Thepelleted seeds were dried overnight at room temperature andwere sown in pots containing field loam. Rice plants raisedfrom bacterized seeds were sprayed twice with P. flu-orescens (108 CFU ml-'), once when the plants were 25 daysold and once 1 day before inoculation with the Sh-R patho-gen.

For field experiments, two locations were used with eightrice cultivars. The effects of bacterization on the suppressionof the development of Sh-R symptoms were studied inreplicated plots raised from bacterized and nonbacterizedseeds.

Plant inoculations. In greenhouse experiments, the riceseedlings (cv. IR 20) raised from bacterized and nonbacte-rized (control) seeds were maintained separately. At booting(the panicle-emerging stage), the middle of the uppermostflag leaf of all plants was inoculated with S. oryzae by thestandard grain inoculum technique. The severity of Sh-Rincidence was recorded by measuring the lesion length.

Similar plant inoculation procedures were followed duringthe three field experiments conducted in 1985 and 1986 attwo locations: the Field Laboratory, Anna University (FL-AU), Madras, India, and the Fredrick Institute for PlantProtection and Toxicology (FIPPAT), Padappai, ChingleputDistrict, Tamil Nadu, India. Eight rice cultivars, i.e., IET

1444, IR 20, IR 36, IR 50, ADT 36, TNAU 840230, TKM 9,and White Ponni, were planted in replicated field plots (3 by3 m during 1985 and 4 by 4 m during 1986 at FL-AU and 2 by2 m at FIPPAT); seedlings were either treated or not treatedwith P. fluorescens isolated from citrus. After the rice plantswere inoculated with S. oryzae, Sh-R incidence was re-corded from 100 randomly selected tillers per plot by using a1-to-8 scale (1).At harvest, the number of normal and infected grains per

tiller, the 1,000-grain weight, and the yield per plot were alsorecorded. For cultivars raised in FL-AU during 1985 and1986, the mean Sh-R disease index was recorded from anaverage of 100 tillers randomly collected from bacterized andnonbacterized plots. Plants of cv. IR 20 raised during a 1986field trial were also evaluated for height and number of tillersper plant when the plants were 60 days old.

RESULTS

Four P. fluorescens strains, isolated from black gram,cowpea, cotton roots, and citrus leaf, and five of the sixstrains isolated from rice belonged to biotype C(III), thenon-levan-forming, nitrifying group of P. fluorescens. Thesixth strain, a levan-forming, nonnitrifying P. fluorescensstrain isolated from rice, belonged to biotype A(I). In tests ofin vitro antibiosis, the strains isolated from rice either werenot antagonistic towards the Sh-R pathogen or were lessantagonistic than the other strains (Table 1). The P.fluorescens strain isolated from cankered citrus leaf showedthe most toxicity towards S. oryzae (Fig. 1). The inhibitionzones induced by this strain had a diameter of 2.5 cm. Thisstrain was used in further greenhouse and field tests.

Bacterization of rice (cv. IR 20) in greenhouse experi-ments with the P. fluorescens strain isolated from citrus ledto the suppression of Sh-R symptoms. In nonbacterized riceseedlings, Sh-R progressed and caused spreading lesions(average diameter, 8.7 cm each), whereas in bacterizedplants restricted and necrotic lesions (average diameter, 4.0cm each) were observed.

Imprints of rice seedlings on KB agar demonstrated thatP. fluorescens was associated with roots of 1- to 3-day-oldrice seedlings. Most of the fluorescence was seen in the root

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2058 SAKTHIVEL AND GNANAMANICKAM

L~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~FIG. 2. Fluorescence photomicrograph showing colonization of

P. fluorescens on seminal root of rice (magnification, x 1,001).

region. In 5- to 7-day-old seedlings, fluorescence was ob-served near the shoot apex, which suggested that P.fluorescens moved along the growing tip of the rice plants.Fluorochrome staining of seminal roots of the rice seedlingsshowed the presence of P. fluorescens on the surfaces ofthese roots. The staining procedure provided a good contrastbetween the fluorescent P. fluorescens cells and the rice rootcells (Fig. 2).

Bacterization of rice with P. fluorescens reduced theincidence of Sh-R in field experiments (Table 2). Bacterizedplants of five rice cultivars examined during 1985 and cv. IR20 examined again during 1986 showed reductions in Sh-Rincidence. The Sh-R reduction was 32 to 42% during 1985and 20% during 1986. The Sh-R-infected grains also were

fewer in earheads (panicles) that emerged from bacterizedrice plants (Table 3). In the field experiments at FL-AU,

TABLE 2. Effect of P. fluorescens treatment on incidenceof Sh-R disease of rice

Yr of Cultivar and Mean disease Reduction instudy" treatment index" disease index (%)

1985 IET 1444U ntreated 8.08Treated 5.34 34C

IR 20Untreated 7.44Treated 4.88 34'

ADT 36Untreated 7.12Treated 4.66 35'

TNAU 840230Untreated 7.38Treated 4.30 42'

IR 36Untreated 6.78Treated 4.62 32"

1986 IR 20Untreated 4.95Treated 3.94 20"

" Studies done at FL-AU.I Average of 100 tillers.C Calculated t value significant at P = 0.01.Calculated t value significant at P = 0.05.

bacterized plants of five rice cultivars showed an 8 to 20%reduction in the number of grains infected (Table 3). In theother location, FIPPAT, bacterized plants of four cultivarsraised during 1986 showed a 2 to 19% reduction in graininfection due to Sh-R (Table 3).

P. fluorescens-treated rice plants were taller and had moretillers. The bacterized cv. IR 20 plants were 11% taller andhad 27% more tillers than did nonbacterized plants (signifi-cant at P = 0.05 based on the Student t test). There was anincrease in the number of grains per tiller, the 1,000-grainweight, and the grain yield per field plot for the bacterizedrice plants (Table 3). This increase was observed in fieldexperiments made at the two locations during 1985 and 1986(Table 3). In FL-AU during 1985 and 1986, yield increasesdue to bacterization varied from 3% in cv. IR 36 to 62% incv. IR 20. In field experiments at FIPPAT during 1986, twoof four cultivars showed enhanced yields of 100% (cv. TKM9) and 160% (cv. White Ponni) due to bacterization, whilethe other two cultivars (IR 50 and IET 1444) did not (Table3).

DISCUSSION

Our data on the in vitro antibiosis of P. fluorescens strainstowards the rice Sh-R pathogen S. oryzae demonstrated thatthis pathogen is sensitive to P. fliuorescens. However, the

TABLE 3. Effect of P. fluorescens-induced suppression ofSh-R disease on yield of rice

No. of Sh-R- 1 0-Yield! IncreaseYr of study Cultivar and infected 1,000-(location) treatment grains/ grain plot in yield

tiller" grains wt (g) (kg) (%)

1985 (FL-AU) IET 1444Untreated 112 16 19 3.6Treated 121 8b 22 4.5 25.0c

IR 20Untreated 108 46 13 2.6Treated 150 26b 17 4.2 62.0b

ADT 36Untreated 96 19 18 2.5Treated 124 11b 20 3.1 24.0c

TNAU 840230U ntreated 89 34 15 2.4Treated 101 12b 20 2.7 13.0d

IR 36U ntreated 111 39 18 3.0Treated 128 16b 20 3.1 3.0d

1986 (FL-AU) IR 20Untreated 104 49 13 4.75Treated 132 30b 18 5.25 10.52d

1986 (FIPPAT) White PonniUntreated 122 34 11 0.30Treated 169 20b 15 0.78 160b

TKM 9Untreated 65 40 19 0.66Treated 94 21b 20 1.33 lOOb

IR 50Untreated 90 21 16 0.87Treated 80 19 17 0.87

IET 1444Untreated 80 31 17 0.77Treated 80 27 18 0.62

"Average of 100 tillers.Calculated t value significant at P = 0.01.Calculated t value significant at P = 0.05.Calculated t value not significant.

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EVALUATION OF P. FLUORESCENS 2059

sensitivity is very variable, and the pathogen exhibits lesssensitivity to those P. fluorescens strains isolated from therice rhizosphere. It is also interesting that three of the sixstrains of P. fluorescens isolated from rice did not show anytoxicity towards S. oryzae, while other strains, isolated fromcitrus, cotton, cowpea, black gram, and rice, showed greatertoxicity. The strain isolated from cankered citrus leaf(biotype c) showed the most toxicity. South Indian P.fluorescens strains have been observed to show in vitroantibiosis toward S. oryzae and other important plant patho-gens, such as Fusarium oxysporum f. sp. vasinfectum (cot-ton wilt pathogen), F. oxysporum f. sp. cubense (panamawilt pathogen of banana), Rhizoctonia solani and Sclerotiumrolfsii (root and stem rot pathogens of peanut), Xantho-monas campestris pv. oryzae (bacterial leaf blight pathogenof rice), and X. campestris pv. citri (citrus canker pathogen)(5, 14, 18).

Antibiosis in vitro does not guarantee antibiosis in vivoand therefore can be misleading if it is used as a criterion forstrain selection. Mew and Rosales (12) observed that theeffect of bacterization of rice on sheath blight control wasnot related to in vitro inhibition of mycelial growth. How-ever, this criterion has been used by researchers (11) forselecting an efficient strain of rhizobacterium. Ganesan andGnanamanickam (5) used this procedure for strain selection,and the use of a selected P. fluorescens strain as a seedinoculant led to the suppression of Sclerotium rolfsii inpeanut. Data from the present study (Tables 2 and 3) alsosuggest that strain selection based on in vitro antibiosis canlead to beneficial results. Perhaps it would be best to selectstrains that have been tested repeatedly both in vitro andunder field conditions before making recommendations tocultivators.What is evident from our data is the variability that can

arise in the beneficial effects of bacterization, even when aselected P. fluorescens strain is used. There was variation inthe reduction of Sh-R incidence in five rice cultivars (Table2). There was also variation in enhanced grain yield of rice ineight rice cultivars (Table 3). The results are indicative of thecomplexity of the biological control and plant growth-promotion phenomenon due to bacterization. Cultivar re-sponses appear to determine the quantum of beneficialeffects due to bacterization.The results presented here, while showing the variability

in the reduction of Sh-R symptoms and enhanced grainyields in rice, clearly demonstrate that substantial reduction(up to 42%) in Sh-R severity and enhanced grain yields (up to160%) are possible, even under different field conditions.Therefore, bacterization of rice, a crop that provides foodfor most people in Asia, may bring about sustained, long-term beneficial effects in disease and crop management.

ACKNOWLEDGMENTS

We thank the Director, Centre of Advanced Study in Botany,University of Madras, Guindy, Madras, India, for providing re-search facilities. We also thank the Director, Fredrick Institute forPlant Protection and Toxicology, Padappai, India, and the Director,Centre for Water Resources, Anna University, Madras, India, forproviding space for field experiments.

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9. Kloepper, J. W., and M. N. Schroth. 1981. Relationship of invitro antibiosis of plant growth-promoting rhizobacteria to plantgrowth and the displacement of root microflora. Phytopathology71:1020-1024.

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16. Suslow, T. V. 1982. Role of root-colonizing bacteria in plantgrowth, p. 187-223. In M. S. Mount and G. H Lacy (ed.),Phytopathogenic prokaryotes, vol. 1. Academic Press, Inc.,New York.

17. Suslow, T. V., and M. N. Schroth. 1982. Rhizobacteria ofsugarbeets: effect of seed application and root colonization on

yield. Phytopathology 72:199-206.18. Unnamalai, N., and S. S. Gnanamanickam. 1984. Pseudomonas

fluorescens is an antagonist to Xanthomonas citri (Hasse) Dye,the incitant of citrus canker. Curr. Sci. (Bangalore) 53:703-704.

19. van Vuurde, J. W. L., and P. E. M. Elenbass. 1978. Use offluorochromes for direct observation of microorganisms associ-ated with wheat roots. Can. J. Microbiol. 24:1272-1275.

20. van Vuurde, J. W. L., and B. Schippers. 1980. Bacterial coloni-zation of seminal wheat roots. Soil Biol. Biochem. 13:559-565.

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