Suppression of Rhizoctonia solani diseases of sugar beet by ...

Journal of Applied Microbiology 2004, 96, 69–75

doi:10.1046/j.1365-2672.2003.02043.x

Suppression of Rhizoctonia solani diseases of sugar beet by antagonistic and plant growth-promoting yeasts K.A. El-Tarabily Department of Biology, Faculty of Science, United Arab Emirates University, Al-Ain, 17551, United Arab Emirates 2002/348: received 3 September 2002, revised 15 April 2003 and accepted 6 May 2003

ABSTRACT K . A . E L - T A R A B I L Y . 2003.

Aims: Isolates of Candida valida, Rhodotorula glutinis and Trichosporon asahii from the rhizosphere of sugar beet in Egypt were examined for their ability to colonize roots, to promote plant growth and to protect sugar beet from Rhizoctonia solani AG-2-2 diseases, under glasshouse conditions. Methods and Results: Root colonization abilities of the three yeast species were tested using the root colonization plate assay and the sand-tube method. In the root colonization plate assay, C. valida and T. asahii colonized 95% of roots after 6 days, whilst Rhod. glutinis colonized 90% of roots after 8 days. Root-colonization abilities of the three yeast species tested by the sand-tube method showed that roots and soils attached to roots of sugar beet seedlings were colonized to different degrees. Population densities showed that the three yeast species were found at all depths of the rhizosphere soil adhering to taproots up to 10 cm, but population densities were significantly (P < 0Æ05) greater in the first 4 cm of the root system compared with other root depths. The three yeast species, applied individually or in combination, significantly (P < 0Æ05) promoted plant growth and reduced damping off, crown and root rots of sugar beet in glasshouse trials. The combination of the three yeasts (which were not inhibitory to each other) resulted in significantly (P < 0Æ05) better biocontrol of diseases and plant growth promotion than plants exposed to individual species. Conclusions: Isolates of C. valida, Rhod. glutinis and T. asahii were capable of colonizing sugar beet roots, promoting growth of sugar beet and protecting the seedlings and mature plants from R. solani diseases. This is the first successful attempt to use yeasts as biocontrol agents against R. solani which causes root diseases. Significance and Impact of the Study: Yeasts were shown to provide significant protection to sugar beet roots against R. solani, a serious soil-borne root pathogen. Yeasts also have the potential to be used as biological fertilizers. Keywords: biological control, growth promotion, rhizosphere competence, soil-borne plant pathogens, yeasts.

INTRODUCTION Large varieties of micro-organisms antagonistic to soil-borne plant pathogens have been reported, but screens for antagonism have focused primarily on bacteria and filamentous fungi (Whipps 2001). There is a paucity of published information with regard to the use of yeasts as biocontrol

Permanent address: Department of Microbiology, Faculty of Science, University of Ain Shams, Cairo, 11566, Egypt (e-mail: [email protected]).

ª 2004 The Society for Applied Microbiology

agents of soil-borne plant pathogens. However, a variety of yeast genera, have been used extensively for the biological control of postharvest diseases of fruits and vegetables (Wilson and Wisniewski 1989; Punja 1997) to protect moulding of stored grains (Petersson et al. 1999) and to control foliar diseases such as powdery mildews (Urquhart and Punja 1997). The mechanisms which were reported to play a significant role in the biocontrol activity of these antagonistic yeasts included competition for space and nutrients (Filonow 1998), production of antifungal diffusible metabolites

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(Walker et al. 1995; Masih et al. 2001), volatile compounds (Payne et al. 2000), cell-wall degrading enzymes such as b-1,3-glucanase (Masih and Paul 2002) and mycoparasitism (Wisniewski et al. 1991). The advantage of using yeasts in biocontrol of foliar and postharvest diseases is caused by the fact that they are the major component of the epiphytic microbial community on the surface of leaves, fruits and vegetables (Wilson and Wisniewski 1989). They are effective as biocontrol agents because they are phenotypically adapted to these niches and are able to effectively colonize and compete for nutrients and space on fruit and leaf surfaces (McLaughlin et al. 1990; Filonow 1998). However, as the biological activities of yeasts on roots, especially of plants producing high levels of stored sugars have not been examined, the rhizosphere of sugar beet was chosen for the present investigation. The role of the rhizosphere micro-organisms in the promotion of plant growth has received increasing attention (Frankenberger and Arshad 1995). Several genera of beneficial free-living bacteria (Glick 1995) and filamentous fungi (Ousley et al. 1994) which inhabit the soil ecosystem, were recognized to have the ability to promote plant growth. Growth promotion effects have been recorded with several biocontrol agents suppressive of root rot pathogens and are associated with the ability of treated plants to compensate for root tissue damage caused by pathogens (Whipps 2001). Postemergence damping off of seedlings, crown and root rots of mature plants, caused by Rhizoctonia solani anastomosis group 2, type 2 (AG-2-2), is a serious disease of sugar beet (Beta vulgaris L.) all over the world (Windels and Nabben 1989). In both upper and lower Nile regions of Egypt, the pathogen causes major yield losses and is becoming an economic threat to commercial growers (Abada 1994). Current disease control measures, including the application of fungicides and/or the use of less susceptible varieties, have not proved to be effective. A biological control method using yeasts as an integrated disease management regime remains to be tested. From previous studies, three promising yeast species namely Candida valida, Rhodotorula glutinis and Trichosporon asahii from a sugar beet rhizosphere which showed different mechanisms of activity against R. solani AG-2-2, were selected for further glasshouse studies. The choice of the three yeast species was based on the ability of C. valida to produce b-1,3-glucanase activity, Rhod. glutinis to produce inhibitory volatiles and T. asahii to produce diffusible antifungal metabolites and to inhibit the growth of R. solani in vitro (K.A. El-Tarabily, unpublished data). Therefore, The objectives of the current study were to determine the ability of C. valida, Rhod. glutinis and T. asahii to colonize roots, suppress diseases caused by R. solani and to promote growth of sugar beet under controlled glasshouse conditions. The three yeast species were included in various

combinations to determine whether biological control could be enhanced by combining test yeast species which show different modes of antagonism to R. solani in vitro.

MATERIALS AND METHODS Yeast isolates The three promising yeast species were identified as C. valida and T. asahii by the Yeast Division, Centraalbureau voor Schimmelcultures (Delft, the Netherlands), and as Rhod. glutinis by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunscweig, Germany). The identification was based on morphological, cultural, physiological and biochemical characteristics. Root colonization assay A preliminary indicator root colonization plate assay (Kortemaa et al. 1994) was carried out in vitro to rapidly indicate whether or not the root exudates of sugar beet (B. vulgaris L.) (cv. Maribo Magna) acting as the sole carbon source, would support the growth of each yeast species. If this was shown, a rhizosphere competence assay using the non-sterile sand tube method was conducted using cycloheximide-resistant strains prepared as described by Suslow and Schroth (1982). Briefly, plates of yeast–malt–peptone– dextrose agar plates (YMPDA) were inoculated with a yeast suspension of sufficient concentration to create a uniform lawn. After 4 h of incubation at 28C, a drop of 0Æ2 ml of sterilized cycloheximide solution (Sigma Chemical Co., St Louis, MO, USA) was placed at the centre of the plates. Resistant strains were isolated from the original plates and re-tested on YMPDA plates containing cycloheximide (150 lg ml)1). In the sand-tube method, sugar beet roots were cut into 2 cm segments after 21 days of growth. The root segments and the rhizosphere soil were studied separately to determine the root-colonization frequencies and population densities of the yeasts in the rhizosphere soils (Kortemaa et al. 1994). Twenty replicates for each yeast isolate were used. Glasshouse in vivo trials Pathogen. A highly virulent isolate of R. solani AG-2-2 isolated from mature, naturally infected sugar beet roots at a farm in Kafr El-Sheikh province, 90 km north of Cairo (Egypt), was used throughout this study. This isolate causes postemergence damping off of seedlings and crown and root rots of mature sugar beet. The pathogen was grown and maintained on potato dextrose agar (PDA) (Difco Laboratories, Detroit, MI, USA) amended with 250 lg ml)1 chloramphenicol (Sigma) and stored at 10C.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 69–75, doi:10.1046/j.1365-2672.2003.02043.x

YEASTS AS BIOCONTROL AGENTS OF RHIZOCTONIA SOLANI

Preparation of R. solani inoculum. The substrate for fungal inoculum was prepared by placing 100 g of millet (Panicum miliaceum L.) seeds to 60 ml of distilled water in 500 ml Erlenmeyer flasks. The flasks were autoclaved at 121C for 30 min on three successive occasions as described by El-Tarabily et al. (1997). This mixture was then inoculated with eight agar plugs (5 mm diameter) from the freshly growing margins of colonies of R. solani under aseptic conditions. The flasks were incubated for 2 weeks and were occasionally shaken to ensure uniformity of growth. Colonized and autoclaved millet seeds served as control material. Preparation of yeast inoculum. Each potentially antagonistic yeast was grown on YMPDA plates for 4 days. For long-term preservation, the cells were removed with a sterile spatula, suspended in 20% glycerol, vortexed for 3 min and stored at )70C. The inoculum for each yeast species was prepared by placing 200 g of moist soya bean bran into 500 ml Erlenmeyer flasks, and autoclaving at 121C for 20 min on three successive days. The substrate was aseptically inoculated with 40 ml of 20% glycerol suspension of each yeast species. The flasks were incubated for 2 weeks and were routinely shaken to ensure uniformity of colonization. Colonized soya bean bran that had been similarly autoclaved served as control material. Soil characteristics. Loamy clay soil was air-dried and passed through a 1-cm mesh sieve prior to use. The chemical characteristics of the soil were analysed as previously described (El-Tarabily et al. 1996). The soil characteristics were: pH, 6Æ8 (in 0Æ01 M CaCl2); electrical conductivity, 1Æ07 dS m)1; organic carbon, 1Æ2%; the following nutrients are expressed in mg kg)1 soil bicarbonate extractable potassium and phosphorus, 352 and 64, respectively; nitrate nitrogen, 37; ammonium nitrogen, 74; sulphate, 85 and iron, 235. Biological control of seedling damping off. Biological control of seedling damping off was determined in the following manner. Soya bean bran colonized with each yeast species was thoroughly dispersed through the air-dried soil by mixing in a cement mixer (0Æ02 g colonized soya bean bran inoculum per gram air-dried soil) 2 weeks before adding the pathogen inoculum. Free-draining polyethylene pots of 30 cm diameter were filled with 8 kg of yeastmodified soil. The pots were then placed in an evaporatively cooled glasshouse maintained at 25 ± 2C, and watered to container capacity twice a week. After 2 weeks, for the treatments which comprised R. solani alone or in combination with yeasts, the contents of each pot were re-mixed with the inoculum consisting of R. solani-infested millet seeds in a cement mixer (0Æ01 g colonized millet seeds inoculum per gram air-dried soil) and repotted.

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In total there were 16 treatment combinations: (1) control (no pathogen), (2) control (R. solani alone), (3) R. solani + C. valida, (4) R. solani + Rhod. glutinis, (5) R. solani + T. asahii, (6) R. solani + C. valida + Rhod. glutinis, (7) R. solani + C. valida + T. asahii, (8) R. solani + Rhod. glutinis + T. asahii, (9) R. solani + C. valida + Rhod. glutinis + T. asahii, (10) C. valida, (11) Rhod. glutinis, (12) T. asahii, (13) C. valida + Rhod. glutinis, (14) C. valida + T. asahii, (15) Rhod. glutinis + T. asahii and (16) C. valida + Rhod. glutinis + T. asahii. Autoclaved infested R. solani millet seeds were used for treatment 1. Sugar beet (B. vulgaris L.) seeds (cv. Maribo Magna) were surface-sterilized in 70% ethyl alcohol for 2 min followed by immersion in 1Æ05% NaOCl (20% household bleach) for 2 min. Surface-sterilized seeds were then washed 10 times with sterile distilled water, dried in a laminar flow cabinet for 10 min and then sown 4 days after soil modification with R. solani (10 per pot) to a depth of 5 mm. The variety Maribo Magna was used as it is highly susceptible to R. solani (Abada 1994). Seedlings were thinned after emergence (ca 5 days after sowing) to six plants in each pot, and each treatment was replicated eight times. The pots were placed in an evaporatively cooled glasshouse and maintained at 25 ± 2C. The free-draining pots were watered daily to container capacity and were fertilized weekly with inorganic liquid fertilizer (Thrive Arthur Yates and Co Limited, Milperra, NSW, Australia) at the manufacturer’s recommended rate. The stand counts were recorded 3 weeks after sowing. Biological control of crown and root rots. Biological control of crown and root rots was determined in the following manner. Sugar beet seeds were surface sterilized as described above and sown in pots containing autoclaved field soil. The pots were placed in an evaporatively cooled glasshouse maintained at 25 ± 2C, watered and fertilized as described above. Five-week-old sugar beet plants were then transplanted into fresh pots containing the same 16 treatment combinations described above. Each treatment was replicated seven times with five plants in each pot. The pots were placed in an evaporatively cooled glasshouse maintained at 25 ± 2C, watered and fertilized as described above. After 9 weeks, taproots were removed, washed and inspected for crown and root rots symptoms. Disease severity was expressed as a disease index rated on a 1–8 scale, modified from Engelkes and Windels (1994): 1, healthy plant (no visible lesions); 2, superficial, arrested dry lesions; 3, < 5%, shallow dry-rot canker at centre of the crown; 4, 5–24% deep dry-rot canker or extensive lateral lesions; 5, 25–49% extensive rot of upper half of taproot; 6, 50–89% of tap root blackened; 7, 90 to < 100% of entire root blackened, except for extreme tip, most foliage dead; 8, 100% completely rotten, dead plant. Plant growth was


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monitored by recording the fresh weight of roots and shoots for the seedlings and mature plants. Statistical analysis All treatments were arranged in a randomized complete block design for all experiments. Population data were transformed into log10 colony forming units (CFU) g)1 dry weight soil. Percentage data were arcsine transformed before ANOVA was carried out. Data were subjected to ANOVA and significant differences between mean values were determined using Fisher’s protected least significant difference test at P ¼ 0Æ05. Superanova (Abacus Concepts, Inc., Berkeley, CA, USA) was used for all analyses.

RESULTS Root colonization assay In the root colonization plate assay, C. valida, and T. asahii colonized 95% of roots by 6 days after radicle emergence, whilst Rhod. glutinis colonized 90% of roots after 8 days. Root-colonization abilities of the three yeast species tested by the sand-tube method showed that roots and soil particles attached to roots of 21-day-old sugar beet seedlings were colonized to different degrees by the three yeast species (Table 1). The three yeast species were found at all depths of the rhizosphere soil adhering to root material, but population densities were significantly (P < 0Æ05) greater in the first 4 cm of the root system compared with other root depths (Table 2).

Table 2 Population numbers in log10 colony-forming units (CFU) for Candida valida, Rhodotorula glutinis and Trichosporon asahii in rhizosphere soil of root segments of sugar beet 21 days after seed treatment Mean log10 CFU g)1 dry soil

Distance from seed (cm)

C. valida

Rhod. glutinis

T. asahii

0–2 2–4 4–6 6–8 8–10

4Æ75 4Æ65 4Æ18 3Æ62 2Æ73

3Æ17 2Æ88 2Æ54 2Æ33 2Æ18

4Æ28 4Æ07 3Æ83 3Æ27 2Æ33

a b c d e

a b c d e

a b c d e

Values with the same letter within a column are not significantly (P > 0Æ05) different according to Fisher’s protected LSD test.

Rhod. glutinis + T. asahii + R. solani) gave the best control and significantly (P < 0Æ05) reduced damping off (Table 3), crown and root rots compared with other treatments (Table 4). Rhizoctonia solani was recovered on PDA amended with chloramphenicol from diseased sugar beet seedlings and mature plants from all modified treatments. The application of yeasts in the presence of the pathogen either singly (treatments 3–5) or in combination (treatments 6–9), or in the absence of the pathogen, either singly (treatments 10–12) or in combination (treatments 13–16) significantly (P < 0Æ05) increased fresh weight of roots and shoots, compared with the controls (treatments 1 and 2) (Tables 3 and 4). Treatment 16 (C. valida + Rhod. glutinis + T. asahii) promoted growth most actively in comparison with other treatments (Tables 3 and 4). DISCUSSION

Glasshouse trials and disease control The application of the three yeasts in pathogen-modified soil individually (treatments 3–5) or in combination (treatments 6–9) significantly (P < 0Æ05) reduced damping off (Table 3), crown and root rots of sugar beet (Table 4) compared with treatment 2 (R. solani alone). Treatment 9 (C. valida +

In this study, the three yeasts C. valida, Rhod. glutinis and T. asahii were demonstrated to be effective biocontrol agents of postemergence damping off of seedlings, crown and root rots of mature sugar beet, caused by R. solani AG-2-2. This is the first record of the use of yeasts as biocontrol agents against a fungal pathogen known to cause root disease. This

Frequency of colonization (%) Distance from seed (cm) 0–2 2–4 4–6 6–8 8–10

C. valida

Rhod. glutinis

T. asahii

Root

Rhizosphere

Root

Rhizosphere

Root

Rhizosphere

100 a 100 a 100 a 100 a 84 b

100 a 100 a 100 a 100 a 91 b

83 75 55 41 23

91 83 61 55 12

100 a 100 a 100 a 75 b 62 c

100 a 100 a 100 a 82 b 71 c

a b c d e

a b c d e

Table 1 Root colonization frequency of Candida valida, Rhodotorula glutinis and Trichosporon asahii in root segments and rhizosphere of sugar beet 21 days after seed treatment

Values with the same letter within a column are not significantly (P > 0Æ05) different according to Fisher’s protected LSD test. ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 69–75, doi:10.1046/j.1365-2672.2003.02043.x


Table 3 Effect of Candida valida, Rhodotorula glutinis and Trichosporon asahii on postemergence damping off of sugar beet caused by Rhizoctonia solani AG-2-2 and on fresh weight of roots and shoots under glasshouse conditions

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Treatments

Plant stand (%)

Root fresh weight (g)

Shoot fresh weight (g)

Control (no R. solani Control (R. solani alone) R. solani + C. valida R. solani + Rhod. glutinis R. solani + T. asahii R. solani + C. valida + Rhod. glutinis R. solani + C. valida + T. asahii R. solani + Rhod. glutinis + T. asahii R. solani + C. valida + Rhod. glutinis + T. asahii C. valida Rhod. glutinis T. asahii C. valida + Rhod. glutinis C. valida + T. asahii Rhod. glutinis + T. asahii C. valida + Rhod. glutinis + T. asahii

98Æ31 11Æ51 79Æ12 75Æ16 81Æ50 83Æ66 86Æ16 84Æ83 91Æ66 97Æ51 98Æ16 99Æ66 99Æ12 98Æ80 98Æ25 97Æ42



h a c b d e f ef g h h h h h h h

b a c c d e f f g i gh hi ij j ij k

b a c c cd def efg de fg hi gh hij ij j ij k

Values with the same letter within a column are not significantly (P > 0Æ05) different according to Fisher’s protected LSD test. Table 4 Effect of Candida valida, Rhodotorula glutinis and Trichosporon asahii on crown and root rots of sugar beet caused by Rhizoctonia solani AG-2-2 and on fresh weight of roots and shoots under glasshouse conditions

Treatments

Disease index

Root fresh weight (g)

Shoot fresh weight (g)

Control (no R. solani) Control (R. solani alone) R. solani + C. valida R. solani + Rhod. glutinis R. solani + T. asahii R. solani + C. valida + Rhod. glutinis R. solani + C. valida + T. asahii R. solani + Rhod. glutinis + T. asahii R. solani + C. valida + Rhod. glutinis + T. asahii C. valida Rhod. glutinis T. asahii C. valida + Rhod. glutinis C. valida + T. asahii Rhod. glutinis + T. asahii C. valida + Rhod. glutinis + T. asahii




a h f g f e d c b a a a a a a a

b a cd c de ef ef f fg gh fg hi ij k jk l

b a cd c cde efg def fgh gh hi ij jk kl m lm n

Values with the same letter within a column are not significantly (P > 0Æ05) different according to Fisher’s protected LSD test.

study also points to the potential of yeasts as biological fertilizers suitable for plant growth promotion. The choice of sugar beet as a host was intentional in the expectation that a plant high in sugar reserves would be an attractive niche for yeasts in general. Earlier reports have indicated dominance of yeasts on fruits (Wilson and Wisniewski 1989; Filonow 1998) and roots rich in sugars (Babeva and Belyanin 1966; Moawad et al. 1986). The biocontrol activity of the yeasts in vivo was positively correlated with in vitro (K.A. El-Tarabily, unpublished data)

inhibition trends. All three yeast species were antagonistic to R. solani when applied individually or in combination, and significantly reduced damping off, crown and root rots under glasshouse conditions. It is interesting that no antagonism was observed among the three yeast species which were able to produce the highest level of disease protection when combined together in the glasshouse trials. Improved control of diseases by combinations of antagonists, in comparison with individual antagonists, may suggest that co-antagonism can occur (Papavizas 1985). Other


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researchers have observed a synergistic effect of two or more antagonists combined into a single treatment, in particular, Mao et al. (1998) found that pepper seeds treated with a combination of Gliocladium virens and Burkholderia cepacia performed better than each antagonist alone in controlling damping off caused by R. solani and Pythium ultimum. In addition to disease suppression, selected yeasts also significantly increased fresh weight of roots and shoots compared with the untreated control. Furthermore, the three yeast species in combination encouraged the greatest plant growth in the glasshouse trials. The growth promotion effect of yeasts may have resulted from these enhanced root and shoot production through the activity of indole-3-acetic acid (IAA) and gibberellic acid (GA3) and possibly other plant growth regulators (PGRs) (Krassilnikov 1963; Tuomi et al. 1993). The isolates of C. valida, Rhod. glutinis and T. asahii used in the present study produced IAA (29Æ5, 24Æ1 and 31Æ7) lg ml)1, and GA3 (6Æ15, 4Æ54 and 7Æ67) lg ml)1, respectively which may affect plant growth (K.A. El-Tarabily, unpublished data). Only a few attempts have been made to use yeasts as biological fertilizers. Abd El-Hafez and Shehata (2001) used a Rhodotorula sp. as a biological fertilizer for tomato plants and reported gains in fruit weight, while the application of Sporobolomyces roseus in Brazil was reported to increase wheat yield by 16–30% (Perondi et al. 1996). Rhizosphere competence appears to be a prerequisite for successful biological control of root diseases, and failure to adequately colonize roots may explain the lack of reliable biological control observed in many studies (Weller 1988). Yeasts have been recorded to occupy the rhizosphere region and to show a rhizosphere effect (Babeva and Belyanin 1966). In the present study, the three test yeast species effectively colonized the root and rhizosphere of sugar beet in turn exhibiting high levels of rhizosphere competence. Rhizosphere competence of yeasts is inadequately addressed in the literature. The results in the present investigation clearly and substantially indicate the need to evaluate the potential place of yeast in the rhizosphere, particularly in relation to its abilities to compete with other micro-organisms in this niche. On the competency of yeasts, ecological studies have been relatively better studied as successful colonizers of the phyllosphere. As R. solani can attack sugar beet at the seedling stage and at maturity, it may be advantageous to establish antagonists in the soil prior to pathogen occurrence or observation. In the present study, the pre-incubation of the yeast antagonists for 2 weeks before addition of the pathogen during glasshouse studies was effective in significantly reducing the diseases. This incubation period may aid in the establishment of introduced yeasts, or enable them to multiply in the soil or to activate the mechanism(s) of antagonism as suggested by Rothrock and Gottlieb (1984).

Although, it is not clear whether these yeast species will be equally successful in protecting roots of plants and plant parts which have relatively less stored sugar material than sugar beet, this study has yielded some interesting information on the biological activity of yeasts in the rhizosphere. Antagonistic activities of yeasts in this niche have unfortunately been relatively neglected in recent studies on the biocontrol of fungal pathogens. This study is expected to bring yeasts into the spectrum of organisms used for the biocontrol of soil-borne plant pathogens. ACKNOWLEDGEMENTS I am greatly indebted to Professor K. Sivasithamparam (University of Western Australia) for his help in the planning of this investigation and in the preparation of the manuscript. Many thanks to Professor Y.A. Youssef (Microbiology Department, Faculty of Science, Ain-Shams University, Cairo, Egypt) for valuable discussions. REFERENCES Abada, K.A. (1994) Fungi causing damping-off and root rot on sugar beet and their biological control with Trichoderma harzianum. Agriculture Ecosystems and Environment 51, 333–337. Abd El-Hafez, A.E. and Shehata, S.F. (2001) Field evaluation of yeasts as a biofertilizer for some vegetable crops. Arab Universities Journal of Agricultural Sciences 9, 169–182. Babeva, I. and Belyanin, A.I. (1966) Yeasts of the rhizosphere. Mikrobiologiya 35, 712–720. El-Tarabily, K.A., Hardy, G.E. St. J., Sivasithamparam, K. and Kurtbo¨ke, I.D. (1996) Microbiological differences between limed and unlimed soils and their relationship with cavity spot disease of carrots (Daucus carota L.) caused by Pythium coloratum in Western Australia. Plant and Soil 183, 279–290. El-Tarabily, K.A., Hardy, G.E. St. J., Sivasithamparam, K., Hussein, A.M. and Kurtbo¨ke, I.D. (1997) The potential for the biological control of cavity spot disease of carrots caused by Pythium coloratum by streptomycete and non-streptomycete actinomycetes in Western Australia. New Phytologist 137, 495–507. Engelkes, C.A. and Windels, C.E. (1994) Relationship of plant age, cultivar, and isolate of Rhizoctonia solani AG-2-2 to sugar beet root and crown rot. Plant Disease 78, 685–689. Filonow, A.B. (1998) Role of competition for sugars by yeasts in the biocontrol of gray mold of apple. Biocontrol Science and Technology 8, 243–256. Frankenberger Jr. and Arshad, M. (1995) Phytohormones in Soils: Microbial Production and Function. New York, NY: Marcel Dekker. Glick, B.R. (1995) The enhancement of plant growth by free-living bacteria. Canadian Journal of Microbiology 41, 109–117. Kortemaa, H., Rita, H., Haahtela, K. and Smolander, A. (1994) Root colonization ability of antagonistic Streptomyces griseoviridis. Plant and Soil 163, 77–83.



Krassilnikov, N.A. (1963) The role of microorganisms in plant life. In Recent Progress in Microbiology ed. Gibbons, N.E. Canada: University of Toronto Press. Mao, W., Lewis, J.A., Lumsden, R.D. and Hebbar, K.P. (1998) Biocontrol of selected soil-borne diseases of tomato and pepper plants. Crop Protection 17, 535–542. Masih, E.I. and Paul, B. (2002) Secretion of beta-1,3-glucanase by the yeast Pichia membranifaciens and its possible role in the biocontrol of Botrytis cinerea causing mold disease of the grapevine. Current Microbiology 44, 391–395. Masih, E.I., Slezack-Deschaumes, S., Marmaras, I., Ait Barka, E., Vernet, G., Charpentier, C., Adholeya, A. and Paul, B. (2001) Characterization of the yeast Pichia membranifaciens and its possible use in the biological control of Botrytis cinerea. FEMS Microbiology Letters 202, 227–232. McLaughlin, R.J., Wisniewski, M.E., Wilson, C.L. and Chalutz, E. (1990) Effect of inoculum concentration and salt solutions on biological control of postharvest diseases of apple with Candida sp. Phytopathology 80, 456–461. Moawad, H., Salem, S.H., Badr-El-Din, S.M., Khater, T. and Iskandar, M. (1986) Yeasts in soils of Egypt. Zentralblatt fu¨r Mikrobiologie 141, 431–435. Ousley, M.A., Lynch, J.M. and Whipps, J.M. (1994) Potential of Trichoderma spp. as consistent plant growth stimulators. Biology and Fertility of Soils 17, 85–90. Papavizas, G.C. (1985) Trichoderma and Gliocladium: biology, ecology and potential for biocontrol. Annual Review of Phytopathology 23, 23–54. Payne, C., Bruce, A. and Staines, H. (2000) Yeast and bacteria as biological control agents against fungal discolouration of Pinus sylvestris blocks in laboratory-based tests and the role of antifungal volatiles. Holzforschung 54, 563–569. Perondi, N.L., Luz, W.C. and Thomas, R. (1996) Microbiological control of Gibberella in wheat. Fitopatologia Brasileira 21, 243–249. Petersson, S., Jonsson, N. and Schnu¨rer, J. (1999) Pichia anomala as a biocontrol agent during storage of high-moisture feed grain

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under airtight conditions. Postharvest Biology and Technology 15, 175–184. Punja, Z.K. (1997) Comparative efficacy of bacteria, fungi, and yeasts as biological control agents for diseases of vegetable crops. Canadian Journal of Plant Pathology 19, 315–323. Rothrock, C.S. and Gottlieb, D. (1984) Role of antibiosis in antagonism of Streptomyces hygroscopicus var. geldanus to Rhizoctonia solani. Canadian Journal of Microbiology 30, 1440–1447. Suslow, T.V. and Schroth, M.N. (1982) Rhizobacteria of sugar beets: effects of seed application and root colonization on yield. Phytopathology 72, 199–206. Tuomi, T., Ilvesoksa, J., Laakso, S. and Rosenqvist, H. (1993) Interaction of abscisic acid and indoel-3-acetic acid producing fungi with salix leaves. Journal of Plant Growth Regulation 12, 149–156. Urquhart, E.J. and Punja, Z.K. (1997) Epiphytic growth and survival of Tilletiopsis pallescens, a potential biological control agent of Sphaerotheca fuliginea, on cucumber leaves. Canadian Journal of Botany 75, 892–901. Walker, G.M., McLeod, A.H. and Hodgson, V.J. (1995) Interactions between killer yeasts and pathogenic fungi. FEMS Microbiology Letters 127, 213–222. Weller, D.M. (1988) Biological control of soil-borne plant pathogens in the rhizosphere with bacteria. Annual Review of Phytopathology 26, 379–407. Whipps, J.M. (2001) Microbial interactions and biocontrol in the rhizosphere. Journal of Experimental Botany 52, 487–511. Wilson, C.L. and Wisniewski, M.E. (1989) Biological control of postharvest diseases of fruits and vegetables: an emerging technology. Annual Review of Phytopathology 27, 425–441. Windels, C.E. and Nabben, D.J. (1989) Characterization and pathogenicity of anastomosis groups of Rhizoctonia solani isolated from Beta vulgaris. Phytopathology 79, 83–88. Wisniewski, M.E., Biles, C., Droby, S., McLaughlin, R., Wilson, C.L. and Chalutz, E. (1991) Mode of action of the postharvest biocontrol yeast, Pichia guilliermondii. 1. Characterization of attachment to Botrytis cinerea. Physiological and Molecular Plant Pathology 39, 245–258.
