Pseudomonas fluorescens, a potential bacterial antagonist to control plant diseases

Abstract

Fluorescent Pseudomonads belong to plant Growth Promoting Rhizobacteria (PGPR), the important group of bacteria that play a major role in the plant growth promotion, induced systemic resistance, biological control of pathogens etc. Many strains of Pseudomonas fluorescens are known to enhance plant growth promotion and reduce severity of various diseases. The efficacy of bacterial antagonists in controlling fungal diseases was often better as alone, and sometimes in combination with fungicides. The present review refers to occurrence, distribution, mechanism, growth requirements of P. fluorescens and diseases controlled by the bacterial antagonist in different agricultural and horticultural crops were discussed. The literature in this review helps in future research programmes that aim to promote P. fluorescens as a potential bio-pesticide for augmentative biological control of many diseases of agriculture and horticultural importance.

Keywords:

• Pseudomonas fluorescens
• strains
• bacterial antagonists
• plant diseases
• biological control
• plant pathogenic fungi

Introduction

Pseudomonas fluorescens encompasses a group of common, nonpathogenic saprophytes that colonize soil, water and plant surface environments. It is a common gram negative, rod-shaped bacterium. As its name implies, it secretes a soluble greenish fluorescent pigment called fluorescein, particularly under conditions of low iron availability. It is an obligate aerobe, except for some strains that can utilize NO₃ as an electron acceptor in place of O₂. It is motile by means of multiple polar flagella. Pseudomonas fluorescens has simple nutritional requirements and grows well in mineral salts media supplemented with any of a large number of carbon sources (Palleroni 1984). Because they are well adapted in soil, P. fluorescens strains are being investigated extensively for use in applications that require the release and survival of bacteria in the soil. Chief among these are biocontrol of pathogens in agriculture and bioremediation of various organic compounds.

Certain members of the P. fluorescens have been shown to be potential agents for the biocontrol which suppress plant diseases by protecting the seeds and roots from fungal infection. They are known to enhance plant growth promotion and reduce severity of many fungal diseases (Hoffland et al. 1996, Wei et al. 1996). This effect is the result of the production of a number of secondary metabolites including antibiotics, siderophores and hydrogen cyanide (O'Sullivan & O'Gara 1992). Hass and Defago (2005) reviewed the mechanisms by which P. fluorescens control pathogenic microorganisms in detail. Competitive exclusion of pathogens as the result of rapid colonization of the rhizosphere by P. fluorescens may also be an important factor in disease control. The present review discusses the occurrence, distribution, growth requirements of P. fluorescens and diseases controlled by the bacterial antagonist in different agricultural and horticultural crops.

Occurrence and distribution

A study during storage of broccoli at 5, 15 and 20°C in Australia (Victoria and New South Wales) indicated P. fluorescens to be predominant. Of the 31 bacterial isolates from phylloplane of Solanum melongena and 20 from Ipomoea batata were characterized, out of that one isolate belonging to P. fluorescens was found to be antagonistic to Macrophomiea phaseolina, Helminthosporium tetramere, Alternaria tenuis and soil-borne Fusarium solani and Sclerotinea rolscii (Atef 2000). Rangeshwaran and Prasad (2000b) collected 300 isolates from different regions of Karnataka. Four isolates belonging to P. fluorescens viz., PDBCAB 2, PDBCAB 19, PDBCAB 29, PDBCAB 30, were most prominent. Total rhizosphere population was higher (log cfu 6.4) for P. fluorescens isolate PDBCAB 29 after 14 days of germination of chickpea. Strains PDBCAB 19 and 30 were able to fully control F. oxysporum f. sp. ciceris. All above four antagonists promoted growth of chickpea. Out of 11 pea cultivars screened at flowering stage (Netherlands), five showed colonization. Pea cv. Twiggy at pod stage showed the highest and most consistent colonization (Elvira-Recuenco & van Vuurde 2000).

In Poland, 94 antagonistic agents from various vegetables and potato were isolated and the most frequent strain was P. fluorescens biovar I. It formed 31.9% of the tomato isolates (Zolobowska & Pospieszny 1999). Mabagala (1999) isolated naturally occurring epiphytic non-pathogenic bacteria from reproductive tissue of various bean genotypes grown in field. Among that one isolate was identified as P. fluorescens which was antagonistic to Xanthomonas axonopodis pv phaceoli in vitro. In Italy, Cirvilleri et al. (1999) collected 182 bacterial isolates from rhizosphere of tomato, lettuce, cauliflower and artichoke grown in 10 commercial green houses. They found that most isolates belonged to P. fluorescens biover I, II, III, V and to P. putida. Zalewska (1999) isolated and tested 138 epiphytic bacteria, out of that 25 isolates belonging to P. fluorescens had limited the growth of Monilia coryli on hazel (Corylus avellana) during 1994–1997 in Poland.

Bonaterra et al. (1998) isolated in Spain several strains of P. fluorescens from a wide range of environments which were antagonistic to brown shoot of pear (Stemphyllium vesicarium). Johnson et al. (1999) observed P. fluorescens up to 10 m from inoculated pear and apple trees as bloom progressed, which suggested that insect vectors were involved in the movement of bacteria. Bacterial population associated with outer florets and the cut surfaces of the stem were ten-fold higher than those associated with inner florets and non cut stems (Padaga et al. 2000).

Klyuchnilov and Kozhevin (1990) in former USSR, found that the most numerous and active population of P. fluorescens were found in the exo-rhizosphere of potato. Hebbar et al. (1991a) studied bacteria associated with sunflower leaves and roots inhibited in vitro growth of Alternaria helianthi, Sclerotium rolfsii, Rhizoctonia solani and Macrophomina phaseolina in France. The root-associated bacteria were identified as P. fluorescens and could be used as seed inoculum to improve plant growth through disease control. Hoflich (1992) isolated 105 bacteria from the rhizosphere of winter wheat; only one P. fluorescens strain (PsIA12) stimulated reproductive growth of winter wheat, winter rape, oil radish, mustard and peas in pot and field experiments. It also inhibited soil borne root pathogens (Gaeumannomyces graminis, Fusarium oxysporum f. sp. pisi and F. solani) in plate tests. Thara and Gnanamanickam (1994) isolated 1757 isolates of bacterial antagonists and identified both fluorescent and non fluorescent groups, 12% inhibited R. solani and 13% (of the remaining total 1366) tested positive for chitinase activity. Tzeng et al. (1994) isolated a total of 151 strains of fluorescent pseudomonads from various crop at different localities in Taiwan. They also found most of the foliar fluorescent pseudomonad strains to be saprophytic.

Mechanism of action

Particular bacterial strains in certain natural environments prevent infections diseases of plant roots. How these bacterial strains achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads (Hass & Defago 2005). The anti-fungal metabolite 2,4-diacetyl phloroglucinol play a major role in the biocontrol capabilities of P. fluorescens (Delany et al. 2000). Karunanithi et al. (2000) observed a native isolate of P. fluorescens producing an antibiotic compound, pyrollnitrin, which inhibited growth of M. phaseolina by producing an inhibition zone of 12 mm. Meena et al. (1999) got a substantial increase in phenyl alanine ammonia lyase activity in rice leaves sprayed with P. fluorescens Pf 1 strain one day after treatment, whereas maximum activity was observed 3 days after treatment. Steijl et al. (1999) working with radish and carnation for control of F. oxysporum f. sp. raphani and F. oxysporum f. sp. dianthi found that fungal infection led to degradation of cell walls in host cells. The lignin component in infected wall was demethoxylated, oxidized and depolymerized. It was also found that infecting radish and carnation with P. fluorescens WCS 417r before pathogen infection led to reduced cell wall degradation. Pseudomonas fluorescens strain CHA0 requires sufficient iron, and iron competition is not a suppressive mechanism in this system. It is thought that HCN production has a role in disease suppression (Kell et al. 1989).

Recently, Imran et al. (2006) reviewed the role of cyanide in controlling root knot disease of tomato. Pseudomonas fluorescens 2-79 produce the antibiotic phenazine-1-carboxylic acid and suppress take-all of wheat caused by Gaeumannomyces graminis var tritici. The antibiotic was isolated only from roots of wheat colonized by strain 2-79 in both growth chamber and field studies in USA (Thomashow et al. 1990). Kell et al. (1992) indicated that the importance of 2,4-diacetyl phloroglucinol production by strain CHA0 (P. fluorescens) is the suppression of soil-borne plant pathogens in the rhizosphere. Borowicz et al. (1992) in Poland observed that the ability of plant growth-promoting fluorescent Pseudomonas inactivate cell wall degrading enzymes of plant pathogenic fungi should be taken into account as an additional mechanism explaining the biocontrol properties of this group. Bacteria isolated in the rhizosphere and roots of maize exhibited varying degrees of antagonism towards F. moniliformae, which was dependent upon the soils from which the bacteria were isolated. It is suggested that antibiotic production rather than siderophores is responsible for anti-fungal activity of antagonistic bacteria (Hebbar et al. 1992). The antagonism was connected to siderophore production on fluorescent Pseudomonas strains and to production of antibiotic like substance in non-fluorescent strains (Cassinelli et al. 1993). Sacherer et al. (1994) at Switzerland found antibiotic metabolites synthesized by P. fluorescens strain CHA0 play an important role in the suppression of root diseases of plants. The production of there metabolites is activated by the global activator gene gacA. Nowak Thompson and Gould (1994) standardized detection methods for several iron binding metabolites (siderophores) of P. fluorescens B10 (JL 3133) using C18 reverse phase HPLC coupled with photodiode array.

Growth studies

Turnbull et al. (2001) studied the bacterial motility in the survival and spread of P. fluorescens SBW25 and attachment and colonization of wheat roots. They constructed motile and non-motile strains and allowed their detection in both soil and water. Although there was no difference between strains in water, the motile strain survived in significantly greater numbers than the non-motile strain after 21 days in soil. The motile strain had a significant advantage in attachment to sterile wheat roots in both non-competitive and competitive studies. They concluded that bacterial motility could contribute to survival in soil and the initial phase of colonization where attachment and movement onto the root surface are important. Pujol et al. (2005) screened P. fluorescens EPS62e for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. The population level of EPS62e after treatment was 7 log CFU (g f.w.)⁻¹, which in turn decreased progressively to 4–5 log CFU (g f.w.)⁻¹ after 17 days and then remained stable until the end of the assay 11 days later. Knee et al. (2001) in the USA purified pea root mucilage which appeared to contain an unusually high amount of material that was similar to arabinogalactin protein. Purified pea mucilage was used as the sole carbon source for growth of P. fluorescens PRA 25.

Ripp et al. (2000) showed that utilizing bioluminescence of a population monitoring tool for lux-based microorganisms, was more effective and precise than standard selective plating techniques, and provided an accurate ecological analysis of P. fluorescens HK44 population dynamics. Srivastava et al. (1999) studied root colonization of wheat in non-sterile soils employing two promising growth promoter strains of P. fluorescens (GRP3 and PRS9). Both strains showed parallel colonization patterns in wheat rhizosphere and rhizoplane. Both strains GRP3 and PRS9 promoted wheat growth in terms of root-shoot length and weight, however, bacterial population decreased towards the root tip. Hase et al. (2000) studied the effect of cucumber roots on the survival pattern of P. fluorescens CHA0-Rif for 22 days in two non sterile soils. They found that soil types had a significant influence on the occurrence of VBNC (viable but non-culturable) cells of CHA0-Rif, although these cells were found in root-associated habitats (i.e., rhizosphere and root tissues) and not in bulk soil.

Gandhi Kumar et al. (2001) found peat, coir pith and talc to be the best substrates for growing P. fluorescens. McGuire (2000) found that surface populations of P. flurorescens were stable between 103 and 104 cfu/cm² on shellacked fruit over 4 months at 13°C. Keinath et al. (2000) showed that zinc and medium dilution were effective for improving genetic stability of other P. fluorescens biocontrol strains obtained from Guana island and Italy. McEldowney (2000) obtained results which suggested that the characteristics of heavy metal (cadmium) accumulation by P. fluorescens H2 was substantially affected by attachment to solid surfaces (glass surface). Nielsen and Sorensen (1999) found 12 isolates of P. fluorescens from barley and sugar beet rhizosphere showing chitinolytic activity in batch cultures when grown in media without exogenous chitin. Nakata et al. (2000) obtained results (radish) which suggested that polysaccharide (Flocculent 1–100 ppm) enhanced adhesion of P. fluorescens S272 cells that might be useful for promoting plant growth through the increased antibiotic activity. Mercier and Lindow (2000) illustrated that plants capable of supporting high bacterial population sizes were proportionally more depleted of leaf surface nutrients than plants with low epiphytic populations. Naseby and Lynch (1999) working with pea (P. sativum var. Montana) in a sandy loam soil of pH 5.4, observed that increasing pH increased the indigenous population of P. fluorescens strain F11₃ compared to reduced pH treatment.

Pseudomonas fluorescens strain CHA0 stored in MS1 clay or in pure vermiculite clay could be re-isolated up to 6 months and survived exposure to 60°C for 24 h in that clay. In clay from MC1, on king's B medium, CHA0 survived less than one month and was killed by exposure to heat (Stutz et al. 1989). The suppressive ability of P. fluorescens strain CHA0 to decrease take-all of wheat (caused by G. graminis var. tritici) and black root of tobacco (caused by Thielaviopsis basicola) was dependent on soil quality and the host-pathogen systems (Wuthrich & Defago 1991). The colonization of potato and radish root system by strain of P. fluorescens was studied in pot culture experiment at UK. Root colonization was extensive but populations were highest on the upper root system and their distribution throughout the root system was greatly affected by environmental factors. Percolation of water through the soil and partial soil sterilization enhanced colonization (Davies & Whitbread 1989). Preliminary analysis of soil samples from Victoria, Australia, indicated P. fluorescens to be a dominant species (63%) (Pascoe & Premier 2000).

Elsherif and Grossmann (1991) found that P. fluorescens applied to seed or root treatment or by placing in the planting hole in pot trials against Plasmodiophora brassicae in Chinese cabbages, it significantly controlled the pathogen. Soil bulk, density and temperature had significant effect on colonization of the rhizosphere by P. fluorescens. Greatest rates of colonization occurred at lower bulk density (0.82g/cm³) and highest temperature (22°C) (Rattray et al. 1993). Thomashow et al. (1990) studied roots of wheat plants grown in steamed soil yielded larger bacterial populations (P. fluorescens 2-79) compared to roots from natural soils. Weidenborner and Kunz (1993) showed that fermentation of P. fluorescens for 24 h at 20°C a Plate Count Broth (PCB; 0.5% casein-peptone, 0.25% yeast extracts, 0.1% dextrose) concentration of 100% enhanced effectively to 93.6% while number of nematodes was reduced to 88.1% in a PCB medium of 50%. Best results were obtained if fermentation was done in a medium containing 0.5% casein-peptone and 0.25% yeast extract. Out of 253 bacterial strains (mostly Pseudomonas spp.) isolated from rhizosphere of plants growing in suppressive soils, 18 strains (50% being P. fluorescens and P. putida) gave more than 35% inhibition of the pathogen, P. ultimum (Cassinelli et al. 1993). Survival of indigenous heterotrophic bacteria (P. fluorescens R₂f, RP₄) depends on the concentration of Cu (IL) and time of incubation (Kozdroj 1994). Casale et al. (1995) found that mulch characteristics which favour healthy growth of citrus and avocado also favour the growth of P. fluorescens.

Control of plant diseases in crops

Cereals

When rice seeds were treated with the formulation of P. fluorescens Pf1 before sowing, at sown and at 30 days, seedlings showed resistance to X. oryzae pv. pryzae, where the disease incidence decreased from 6.8–1.2 (Vidhyasekaran et al. 2001). In a field trial, Karpagavalli et al. (2002) investigated the complementary effect of silica (6 t lignite fly ash (LFA)/ha) along with a foliar spray of the biological control agent, P. fluorescens, on the blast [Magnaporthe grisea] incidence of rice cultivars IR50 and White Ponni. Foliar spray of P. fluorescens, in addition to LFA and 45 kg potash/ha significantly reduced the rice blast incidence and increased crop yield. When seeds were treated with P. fluorescens strain PfALR2, Mishra and Sinha (2000) got increased seed germination of rice from 26.3–52.6%. Two P. fluorescens strains, viz. PF1 and FP7, inhibited the mycelial growth of sheath blight fungus R. solani and increased the seedling vigour of rice plants and yield under green house and field conditions. Pseudomonas treatment of rice cv. IR50 led to induction of systematic resistance against R. solani as a result of increase in chitinase and peroxidase activity (Nandakumar et al. 2001).

Field trials with rice cultivars Co 43, ADT 36 and ADT 38 were conducted during Kuruvai (June–September), Samba (August–January) and Navarai (January–April) seasons, to evaluate the efficacy of P. fluorescens strain Pf-1, in controlling Hirschmanniella gracilis. This was done by giving seed treatment (10 g/kg) and nursery soil application either with or without soil application of carbofuron 3g @ 1.3g ai./m². Among the treatments, 10 g/kg of the biocontrol agent was found to be superior to all other treatments. Maximum bacterial colonization and nematode suppression along with rice yield/increase (13%) was observed in this treatment (Ramakrishnan et al. 1998). Rajbir Singh and Sinha (2005) studied the effect of P. fluorescens strains 1 and 5 against sheath blight, R. solani on rice under glasshouse conditions. They found that P. fluorescens of higher rate, i.e., 8 g/l was highly effective in reducing disease severity (60.0%) and incidence (35.6%) and increasing grain yield (33.8%) and 1000-grain weight (12.9%).

In wheat (Triticum aestivum L.), two strains of Pseudomonas GRP3 and PRS9 promoted wheat growth in terms of root-shoot length and weight. Kita et al. (2004) found P. fluorescens strain PSR21 to be a good measure for a wheat seed treatment and later, during the spring, spraying resulted with a noticeable decrease of the average degree of culms damage. Wang-Ping et al. (1999) observed that P. fluorescens biovar I and III successfully reduced the disease incidence (Helminthosporium sativum) and increased plant height and dry weight. Generally, bacterial (P. fluorescens) population decreased towards the root tip (Srivastava et al. 1999). In a glasshouse experiment, the effects of different fly ash concentrations (0, 20, 40%) and soil microorganisms (P. fluorescens, Azotobacter chroococcum, Glomus mosseae and Aspergillus awamori) on the plant growth, photosynthetic pigments and leaf blight of wheat caused by A. triticina were tested. Glomus mosseae caused the greatest increase in plant growth and photosynthetic pigments and greater reduction in the percentage of infected leaf area followed by P. fluorescens (Siddiqui Singh 2005). The bacteriarization of wheat weeds with P. fluorescens strain WCS 417 in the preceding year had limited the natural build up of G. graminis. Only 6% white heads had developed in these fields and yield was increased significantly (Lamers et al. 1988). McManus et al. (1993) found that common bunt disease (Tilletia laevis) was reduced by 65 and 50% during consecutive seasons when wheat seeds and 2-week-old seedling were treated with Pf2-79r. Whereas, the strain D7 suppressed germination of seeds and reduced root and shoot growth of downy brome (Bromus tectorum) in agar diffusion assay. The inhibition was complete at concentrations as low as 1 mg total dry matter/liter agar. Also, the active fraction inhibited the plant pathogenic fungus Gaemannomyces graminis var. tritici (Gurusiddaiah et al. 1994) on wheat.

Seeds of ragi (Elengine coracana var. paiyur 1) treated with P. fluorescens against blast disease caused by Pyricularia grisca indicated that it was not significantly better than fungicidal treatments (Vanitha 1998). Umesha et al. (1998) carried out an experiment under greenhouse and field conditions in Karnataka, India. Where they treated seeds of pearl millet (Pennisetum glaucum) with P. fluorescens formulated in talc powder which increased seedling vigour and inhibited sporulation of the downy mildew pathogen. P. fluorescens controlled downy mildew by both seed treatment and foliar application, but efficacy was significantly higher when seed treatment was followed by a foliar application.

Pulses

P. fluorescens strain RPB14 showed potential biological control activity against major diseases of Phaseolus vulgaris cv. Baspa both in vitro and under field conditions (Himachal Pradesh, India). Two foliar applications of RPB14 and a single spray of carbendazim (0.05%) were best in protecting from both diseases by 59.6% (floury leaf spot) and 51% (fuscous blight) and in increasing the seed yield by 81.3% over the control (Mondal 2004). Pseudomonas fluorescens strain Pf1, effectively inhibited the mycelial growth of Macrophomira phaseolina, the pathogen causing dry root rot in Black gram cv.Co5 application of strain Pf1 (10 g/kg seed) followed by soil application (2.5 kg/ha) against root rot effectively supported higher plant growth, better Rhizobium nodulation and grain yield (Jayashree et al. 2000). Field trials in Vigna mungo to control root rot disease complex caused by Macrophomina phaseolina and cyst nematode, Heterodera cajani was carried out at Coimbatore. Pseudomonas fluorescens was applied as seed treatment (2 g/kg seed) and the results showed less root rot incidence and nematode population along with increased pod yield (Latha et al. 2000). Siddiqui et al. (1998) used P. fluorescens alone or in combination with pesticides to control wilt disease complex of Pigeon pea, H. cajani. Pseudomonas fluorescens alone increased plant growth, nodulation, phosphorus content and decreased nematode multiplication and wilting in infected plants. Shaid Ahamad et al. (2000) conducted field trials at Kanpur, India for controlling root rot disease (R. bataticola) in Chick pea cv. C235 using bacterial antagonist P. fluorescens at 500 g/ha. It gave some control compared to untreated control when given as soil inoculation plus seed treatment.

In chickpea P. fluorescens (PDBCAB 2) treated plots exhibited low root-rot (4.4%). However, at day 60 lowest root-rot incidence (5%) was recorded in strain PDBCAB 2 treated plots and highest root-rot incidence (13.9%) was observed in control (Rangeshwaran et al. 2001). Plant stand and fresh weight of navy beans (Phaseolus vulgaris) were survived well on the P. fluorescens treated seeds for more than 10 weeks in storage (Tu & Zheng 1997).

Fruits

In grape vine cv. Muscut Hamburg, a talc formulation of P. fluorescens (15×10⁸ cfu) was applied around 15 cm soil to root knot infested vines. Depth is the basic one at the time of pruning in July, 1996 at Coimatore, India. They found all three levels significantly reduced the severity of root knot infection in roots. Yield of grape increased under P. fluorescens treatment which ranged from 45% at dosage 1g/vine to 166% at 4g/vine (Shanthi et al. 1998). In sweet orange (Citrus sinensis) and lemon (C. limon), application of talc-based formulation of P. fluorescens strain Pf1 40 g/tree, retarded multiplication of T. semipenetrans significantly in field trials at Bhavanisagar, Tamilnadu. Combining this treatment with carbofuron 2g ai./tree further enhanced the suppression of the nematode. The recovery in the declining trees was also accompanied by a significant decrease in the nematode parasites in roots (Shanthi et al. 1999). Kucheryava et al. (1999), proposed five strains of P. fluorescens as biocontrol agents for the control of Venturian equalis, the causal agent of apple scab disease in apple at North Germany. They isolated and characterized epiphytic bacteria from phyllosphere of apple. In blueberry (Silva et al. 2000) (Vaccinuim corymbosum cv. Blue crop), P. fluorescens strain Pf 5 increased leaf area and number and shoot and dry weight in pasteurized root. PRA 25 strain increased copper and phosphoras uptake.

Pseudomonas fluorescens A506 suppressed the epiphytic growth of A. avenae subsp. citrulli when applied to attached watermelon blossoms 5 h prior to inoculation (Fessehaie & Walcott 2005). Application of P. fluorescens strains 558 significantly reduced anthracnose in mango caused by Colletotrichum gloeosperoides when the fruits were inoculated by the antagonist (Koomen & Jeffries 1993) in the UK. Strain Pfcp protected banana plants from wilt disease caused by P. solanacearum up to 50% in the greenhouse and in the field (Anuratha & Gnanamanickam 1990) at Madras, India. Wilson and Lindow (1993) concluded that strain A506 probably prevents fire blight infection of pear in the field by preventing epiphytic build-up of pathogen inoculum on pistils and by inhibiting the growth of inoculum deposited on nectarines. Whereas, strains 1-1-4 provided more than 95% crown gall disease control in peaches at China (Zhang et al. 1991). Pseudomonas fluorescens provided 0–60% disease control of apricot and peach cutting inoculated with Leucostoma cinctum, and treatment of cutting with the bacteria before inoculation provided better control (Rozsnyay et al. 1992).

Vegetables

Keinath et al. (2000) isolated 13 bacterial strains from field in the southern USA having damping off history. The seeds of snap bean were treated in bulk with P. fluorescns C 200 and BD4-13. Analysis of variance of percent plant stand at 28 day after sowing revealed highly significant (p<0.01) effects of location and treatment in 1996, 1997 and 1998. Data collected for 2 years indicated that no biological seed treatment significantly affected plant stand. In Phaseolus vulgaris, seed-borne Colletotrichum lindmuthianum was controlled by P. fluorescens but it was only next to T. viride in efficacy (Ravi et al. 1999). Naseby et al. (2001) assessed four strains of P. fluorescens against P. ultimum in pea and found the disease incidence to reduce, seed bactrerialization of pea by P. fluorescens in combination with a aerial spray of their cell suspensions or neemazol (product of neem) at different concentration were tried under field conditions at UP, India. The spray combination increased dry weight of aerial parts, number of nodes and pods and seed weight of pea. Colonization of rhizosphere was fairly good in strain Pf 5 (Singh et al. 2000).

Khan and Akram (2000) achieved enhancement of plant growth and yield in tomato from nematode- (M. incognita) and fungus- (F. oxysporum) infected plants by treating them with P. fluorescens in field trials at Aligarh, India. Thiribhuvanamala et al. (1999) got significantly reduced mycelial growth and sclerotial production of Sclerotium rolfsii (in vitro), the causal agent of stem rot in tomato. Park Chang Seuk et al. (2001) confirmed that the root dipping of tomato seedlings in strain B16 cell suspension (109 cfu/ml) when transplanting was sufficient to suppress bacterial wilt till fruit bearing. P. fluorescens was found effective in reducing (54.30%) the fruit rot disease of tomato (Hegde & Anahosur 2001). Dekkers et al. (2000) found P. fluorescens WCS 365 to be an efficient root colonizer when tomato seeds were inoculate with this strain to control root rot, F. oxysporum f. sp. rakius-lycopersici. Hultberg et al. (2000) found P. fluorescen Pf 5-014 and its mutant strain 5-214 to significantly reduce fungal population (damping off by P. ultimum) in in vitro cultures containing both high and low inoculums. Spraying tomato cv. PKM-1 with P. fluorescens, 48 h after inoculation with A. solani reduced leaf blight in tomato by 15–38% (Whistler et al. 2000). Hanna et al. (1999) carried out bioassay and greenhouse tests to control M. incognita in tomato. When broth culture (150 ml) was adjusted to 10⁸ c.f.u/ml P. fluorescens showed the most nematicidal activity against hatched juveniles and adults of M. incognita. They found that increased concentrations (10⁵ log and 5×10⁸ cfu/ml.) increased mortality. Tomato cv. Solan Gola is susceptible to wilt (R. solanacearum); least wilt incidence (27.77 and 22.22%) was observed when NaNO₃ was combined with P. fluorescens during 1998 and 1999, respectively, and the wilt population was reduced, when compared to the control (Pradeep Kumar et al. 2003). In Algeria (Botero Ospina & Aranzazu Hernandez 2000) got significant antagonism against F. oxysporum f. sp. lycopersici in tomato by P. fluorescens, in pot culture trials (Manoranjitham et al. 2000a), and it also decreased disease population of P. appomidermatum, the causal agent for damping off on tomato. It also increased shoot, root length and dry weight of seedling (Manoranjitham & Prakasam 1999).

Two strains of P. fluorescens, B1 and B2, were evaluated in a growth chamber and in the field against R. solani in potato and in lettuce. The greatest disease suppression effect on potato was achieved by strain B1 (37%), followed by B2 (33%), whereas the marketable tuber yield increased up to 12% (B1) and 6% (B2) (Grosch et al. 2005).In potato cv. Russet Burbank inoculated with P. fluorescens bv V and bv I, in the first year, P. fluorescens S22: T: 04 at 1×10⁸ cfu/ml decreased dry root rot caused by G. pulicaris by 19%. In the second year, P. fluorescens P.22:Y:05 at 4×10⁸ cfu/ml reduced severity of disease by 25% (Schisler et al. 2000). Manoranjitham et al. (2000b) treated chili seeds with T. viridae (4g kg⁻¹) + P. fluorescens (5 g kg⁻¹) and found 7.0 and 12.5% pre- and post-damping off respectively as against 27.5% and 54.75% in control. There increased root, shoot length and dry weight of chili seedling and decreased the population of P. apnanidermatum from 16.75×10² cfu g⁻¹ at the time of sowing to 13.41×10² cfu g⁻¹ at 20 days after sowing compared to 17.5×10² cfu⁻¹ and 17.08×10² cfu⁻¹ in control. Manoranjitham and Prakasam (2000) continuing their studies, found that seed treatment of chili with talc-based formulation of P. fluorescens effectively reduced damping off and the combination with T. viridaae at 5 g/kg recorded 31.65, 66.6 and 37.58% increase in root, shoot length and dry matter production over control respectively. Sharifi-Tehrani and Omati (1999) used two strains of P. fluorescens in vitro and in vivo to control damping off in bell pepper. They inoculated 12.5 mg of fungal inoculum and about 10⁹ cfu of bacteria (P. fluorescens)/g of soil prior to filling pots and transplanting of seedlings. When results were analysed 30 days after transplanting it was found that P. fluorescens strains provided some protection but the level of protection was less than those provided by B. subtilis.

Rajappan and Ramaraj (1999), working on cauliflower to control wilt disease caused by F. moniliformae, found that soil application of talc-based formulation of P. fluorescens effectively controlled the disease under field conditions. Control of Pythium root rot in cucumber was done in hydrponic systems by Zheng et al. (2000) where, P. fluorescens reduced mucilage and root discoloration more effectively than P. chlororaphis but did not significantly promote marketable yield (on a fresh mass basis). Brovko and Brovko (2000) in Russia, estimated cucumber yield loss ranging from 10–15% to 91–95% due to Fusarium root rot caused by Fusarium species. Treatment with Rhizoplan (P. fluorescens) diluted to 1:50 with water, improved disease resistance and increased yield by 8.5–16.2%.

When sugar beet seeds were inoculated with P. fluorescens DR54 in soil microorganisms, an improvement in plant emergence was found after 7 days. Strain DR54 reduced mycelial density, oospore formation and intracellular activity of P. ultimum (Thrane et al. 2000). In lentil (Lens culinaris) var. PDL 2 and Sehore 74-3, field experiments were conducted to control lentil wilt (F. oxysporum f.sp. lintis) during 1995–1996 and 1996–1997 (De & Chaudhary 1999). Pseudomonas fluorescens+T. viridae and carboxin reduced wilt disease by 65% and increased yield by 229%. In commercial greenhouse trials, P. fluorescens strain WCS 374 suppressed Fusarium wilt and increased radish yield (Leeman et al. 1991). Two isolates of P. fluorescens (PF1 and PF2) inhibited P. solanacearum, which causes bacterial wilt of potato in vitro (Shekhawat et al. 1993). PF2 failed to reduce wilt incidence. Stanlit et al. (1994) found that the post harvest rooting of Dutch white cabbage was reduced by post-harvest treatment with P. fluorescens strains CL 42, CL 66, CL 82 in three storage trials carried out in an experimental cold store. Pseudomonas fluorescens gave a consistently high level of control of damping off (Pythium debaryanum and P. ultimum) in beet root at both 2 and 4 weeks (Dodd & Stewart 1992) at New Zealand. And it is found effective against the five pathogenic seed-borne fungi of okra and increased the percentage of germination, seedling vigour and reduced seedling mortality (Gurjar et al. 2004). In peas (Pisum sativum), P. fluorescens used without captan was very effective, increasing both emergence and yield of 33% when compared to captan alone (Parke et al. 1991).

Flowers

Carnation plants grown in hydroponics solution inoculated with P. fluorescens showed a significant reduction in the number of plants infected with F. oxysporum, whereas strain M24 (Collected from 15-year-old avocado roots at Queensland) gave significant protection of roots of Jacaranda acutifolia from infection by Phytophthora cinnamomi when the fungal inoculum level was 0.0016 g colonized bran/sand per gram (Sorokina et al. 1999).

Oil crops

As biological control agent, P. fluorescens have been shown to have beneficial effect on plant growth and health. Shanmugam et al. (2002) conducted a study to test the effect of P. fluorescens Pf 1 on co-inoculation in peanut [groundnut] to control root rot, a severe soil-borne disease caused by Macrophomina phaseolina. Strain Pf 1 resulted in significantly inhibition of M. phaseolina mycelial growth under in vitro conditions. Leaf shoot disease caused in groundnut by Cercosporidium personatum was reduced when the groundnut seeds were treated with P. fluorescens Pf 1, along with a foliar spray at seedling stage. The talc-based powder formulation effectively controlled the disease under field condition in Madurai in 1999. The pod yield increased and maximum disease protection was observed 30 days after sowing by seed treatment with P. fluorescens (Meena et al. 2000a). Meena et al. (2000b) continuing their studies on groundnut found that foliar application of P. fluorescens strain Pf1 significantly controlled late leaf spot and rust (Puccinia arachis) in greenhouse conditions. An increase in activity of phenyl alanine ammonia lyase one day after application of bacterial antagonist was seen, and maximum activity was observed 3 days after treatment. Vanitha (1998) used 6 carriers (farm yard manure, gobar gas effluent, peat soil, groundnut shell, municipal compost and coconut coir pith compost) for soil application of P. fluorescens against groundnut collar rot incidence. The lowest disease incidence (23.33%) was found in peat soil inoculums followed by farm yard manure and goober gas effluent inocula both at 30% as against 85% in control. In Dharwad (Patil et al. 1998), P. fluorescens strain FDP-15, isolated from groundnut roots was most efficient and ecologically fit strain. This strain improved seed germination, nodulation, dry weight and pod yield as well as protected plants from sclerotial infection compared with captan. FDP15 increased seedling emergence by 16%, nodulation 18%, dry weight 40%, total pod yield 65% and resulted in 18% greater survival of plants up to harvest.

In safflower (Prashanthi et al. 2000), P. fluorescens strain Pf1 produced the highest inhibition zone of Macrophomina phaseolina, the causal agent for root rot of safflower, in sesame cv. TMV6, and strain Pf1 effectively inhibited the mycelial growth of M. phaseolina, the pathogen causing dry root rot in sesame (Jayashree et al. 2000). In sunflower, the highest sclerotium root/collar rot disease suppression was exhibited by P. fluorescens strain PDCAB 2. They also found that relying on the greenhouse test was better than the laboratory test (Rangeshwaran & Prasad 2000a). A positive correlation between in situ tests and field trial demonstrated that significant protection of sunflower from Sclerotini sclerotirum could be obtained by seed bacteriarization with P. fluorescens which gave a satisfactory level of root colonization and biocontrol was obtained in three different soil types (Expert & Digat 1995). Hebbar et al. (1991b) found that the antagonistic bacteria (P. fluorescens) associated with sunflower roots and leaves when used as a seed inoculum in the presence of C. rolfsii increased seedling emergence by 28%. It also improved plant yield through disease suppression.

Cotton

In cotton (Mondal et al. 2000), P. fluorescens strains CRb 26, CRb 39, protected plants from bacterial blight caused by X. axonotrodis pv. Malvacearum (Xam R-32). CRb 26 produced four major phenolic compounds I, II, III, IV. Compounds I and IV were fluorescent. Compounds II and IV completely inhibited growth of Xam R-32 at 200 and 100 µg/ml in vitro. Demir et al. (1999) isolated 128 isolates of fluorescent Pseudomonads from healthy cotton seedlings and rhizosphere soils and tested against Rhizoctonia solani. P. fluorescens (Gh/R 1810) was the most effective strain resulting in 16.36% greater emergence and 57.94% greater survival of cotton seedlings. Hagedorn et al. (1990) found that application of P. fluorescens strain EG1053 provided larger plant stands and reduced seedling disease symptoms (caused by P. ultimum and R. solani) on surviving plants of cotton in both potting mix with amended pathogens and naturally infected cotton soils.

Other crops

P. fluorescens was effective in reducing R. solani disease incidence of Phyllanthus niruri in greenhouse conditions (Ayyanar Kamalakannan et al. 2004). In forest nurseries at Haruana, India, P. fluorescens was more effective against damping off than B. subtilis (Kaushik et al. 2000). Bora et al. (2000) found P. fluorescens M15 to control brown plaster disease in mushroom at the rate of 86.6%. Anith et al. (2000) found seed treatment of ginger with P. fluorescens strain EM85 along with solarization decreased wilt (R. solanacearum) incidence to 7.42% and increased yield to 29.43 t/ha compared to 19.51 t/ha in control.

Pseudomonas tolassi is a major cause of mushroom spoilage in Australia. Growth of P. tolassi was greatly reduced by prior colonization of the mushroom beds by P. fluorescens (Healey & Harvey 1989). In cocoa, P. fluorescens isolates from the surface of healthy cocoa were antagonistic to P. palmivera (in vitro and in the field) and were more effective than copper oxide or chlorothalonil in controlling black pod (Galindo et al. 1992). Nicotiana flutinosa and 2 cultivars of N. tobacum were grown in autoclaved natural soil previously inoculated with P. fluorescens strain CHA0. After six weeks, all tested plants showed resistance in leaves to infection with Tobacco Necrosis Virus (TNV) to the some extent as plants previously immunized with TNV. Root colonization of tobacco plants with strain CHA0, as well as leaf infection with TNV, caused an increase in salicylic acid in leaves (Maurhofer et al. 1994).

Berg et al. (2000) found that P. fluorescens isolate P6 and P10 were found to be antagonistic to Verticillium wilt pathogen and increased the yield from 117–344% (P10) in greenhouse trials and 113–247% (P6) in field trials. Pseudomonas fluorescens, isolates from rhizosphere of winter rape, was antagonistic to pathogenic and saprophytic fungi on rape and flax and protected germinating plants against infections by Phoma lingam (Leptosphaeria maculans), F. acenaceam (Gibberella avanacea), respectively. The antagonistic effect of P. fluorescens+nutrient medium was increased by 20% with treatment with the bacterium alone. Leptosphaeria maculans was suppressed more than G. avenacea. The bacterium was non-pathogenic to rape and flax and tolerant of all herbicides tested (Novotna 1990).

Conclusions

Environmental and consumer concerns have focused interest on the development of biological control agents as an alternative, environmentally-friendly strategy for the protection of agricultural and horticultural crops against phyllopathogens (Dunne et al. 1998). Pseudomonas fluorescens is one such proven biological control agent. Many success reports by several scientists around the world have described different Pseudomonas strains able to significantly control a number of fungal, bacterial and nematode diseases in cereals, horticultural crops, oil seeds and others. The efficacy of bacterial antagonism in controlling diseases was often better than with fungicides. However, the bacterial antagonism in combination with fungicides sometimes improved efficacy in controlling diseases. Besides disease control, treatments also improved seedling health and yields of crops. Peat soil was found to be the best substrate followed by farmyard manure and gobar gas for colonization of P. fluorescens. Polysaccharides enhanced the adhesion of P. fluorescens S272 which promoted plant growth through increased antibiotic activity. The present review contributes to future research programmes that aim to promote P. fluorescens as a potential bio-pesticide for augmentative biological control of many diseases of agriculture and horticultural importance. However, a better understanding of the factors involved, the signalling interaction among antagonist, pathogen, soil and plants, are yet to be revealed to promote the biocontrol agents as wide applicable bio-pesticides in future.

The authors are grateful to Dr S. D. Shikamani, Director, Indian Institute of Horticultural Research, Bangalore and also thankful to Dr Poonam Sinha, (IIHR) Senior Scientist, for her help in literature selection.