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Biocontrol of Rhizoctonia solani and Pythium ultimum on Capsicum by Trichoderma koningii in potting medium
Microbiol. Res. (1999) 154, 131-135 http://www.urbanfischer.de/journals/microbiolres ©
Urban & Fischer Verlag
Biocontrol of Rhizoctonia solani and Pythium ultimum on Capsicum by Trichoderma koningii in potting medium A. R. Harris
CSIRO Division of Soils, and Cooperative Research Centre for Soil and Land Management, Private Bag No.2, Glen Osmond, S.A. 5064, Australia Accepted : June 17, 1999
Abstract Two isolates of Trichoderma koningii were evaluated for efficacy in control of damping-off diseases in seedlings of Capsicum annuum grown in pasteurized potting medium in a glasshouse. A selected isolate of binucleate Rhizoctonia and two fungicides were also included as standards for control of Rhizoctonia solani and Pythium ultimum var. sporangiiferum. Both isolates of T. koningii reduced seedling death caused by R. solani in one of two experiments, and by P. u. sporangiiferum in two of three experiments. Neither isolate of T. koningii suppressed damping-off caused by either pathogen as consistently as the binucleate Rhizoctonia or fungicides. The implications of these results for commercial disease management are discussed. Key words: Trichoderma koningii - binucleate Rhizoctonia Pythium ultimum var. sporangiiferum - Rhizoctonia solani Capsicum annuum - biological control- damping-off - potting medium - fungicides
(Stephens et al., 1982). In previous papers, we have reported on the efficacy of selected isolates of soil bacteria and binucleate Rhizoctonia, including BNR1, for suppression of these pathogenic fungi (Harris et al., 1993 b, 1994 a, b, c; Harris and Adkins, 1999), and plant growth promotion (Harris, 1999). Trichoderma spp. are important biological control agents (Papavizas, 1985), and several products that contain Trichoderma spp. are sold to control a variety of plant pathogens (Fravel et al., 1996). Isolates of T. koningii Oudem. have been shown to be antagonistic to soil-borne pathogenic fungi (Simon, 1989), including R. solani AG 8, Pythium middletonii and Phytophthora cinnamomi (Simon et ai., 1988). The aim of this research was to determine whether T. koningii isolates 7a and 7c have potential for control of damping-off diseases in bedding plants, and to compare their efficacy with one of our selected binucleate Rhizoctonia isolates, BNRl.
Introduction
Materials and methods
The nursery industry needs an economical, environmentally safe biological product to control seedling damping-off diseases caused by Pythium spp. and Rhizoctonia solani KUhn (teleomorph = Thanatephorus cucumeris (Frank) Donk) (Stephens and Powell, 1982; Cline et al., 1988; Harris, 1995). In the Ohio bedding plant industry, damping-off attributed to Rhizoctonia is caused primarily by R. soiani anastomosis group (AG) 4
T. koningii isolates 7a and 7c were isolated originally from wheat soil in Western Australia that was suppressive to Gaeumannomyces graminis var. tritici (Simon, 1989). Pythium ultimum var. sporangiiferum Drechsler isolate 2, R. solani isolate DIBI (AG 4) and binucleate Rhizoctonia isolate BNRI (AG-K) were isolated from plant nurseries around Adelaide, South Australia (Harris et al., 1993 a, 1993 b). These fungi were cultured on sterilized organic substrates and added to pasteurized potting medium (pH(OOlMCaC12) = 5.6) in six-celled plastic punnets (seedling trays) (Harris et at., 1993a). R. solani on wheat bran was mixed with potting medium, and 7 cm3 of this mix was added to the bottom of
Corresponding author: A. R. Harris, Present address: Australian Quarantine and Inspection Service, GPO Box 858, Canberra, A.c.T. 2601, Australia. (E-mail: [email protected]) 0944-5013/99/154/02-131
$12.00/0
Microbiol. Res. 154 (1999) 2
131
each punnet cell. Punnets then were filled with either uninfested potting medium or potting medium infested with BNRI and/or P. u. sporangiiferum. BNRI and P. u. sporangiiferum were cultured on pulverized rice hulls (Harris et a1., 1993 a). Fungal doses are given below. Six seeds of Capsicum annuum L. 'Green Giant' (capsicum, syn. bell pepper) were sown near the top of the potting medium in each punnet cell, and covered with 5 -1 0 mm (approx. 7 cm3) of sterilized, washed coarse sand. The biological control efficacy of the selected fungal antagonists was compared with the fungicides quintozene and propamocarb, which are registered in Australia for control of Rhizoctonia and Pythium respectively in some seedlings. Quintozene (PCNB) (Terraclor, Uniroyal Australia) was suspended in deionized water at 5 g 1-1, and propamocarb (Previcur, Schering AG) at 1.78 mIl-I. 2.25 ml of each suspension was pipetted onto the soil surface in each punnet cell to give applied doses of quintozene of 8.4 mg active ingedient (a.i.) per punnet cell and propamocarb of 2.4 mg a.i. per punnet cell, which approximate the recommended commercial rates. Punnets were arranged in randomized complete block designs with four blocks in a glasshouse at 25°C (± 5°C), and watered twice weekly with a nutrient solution (Harris et az', 1993b). When most seedlings had four true leaves and damping-off had ceased (3-4 weeks), the surviving and collapsed seedlings were counted separately, the standing seedlings were excised at soil level, and the tops were dried at 60°C and then weighed. Root samples from randomly selected punnet cells containing each fungus were washed, plated onto water
agar (Oxoid No.3, Unipath Ltd., Basingstoke, Hampshire, England) or 114-strength potato dextrose agar (Difco Laboratories, Detroit, Mich.), and incubated at 25°C to confirm that the added fungal genera were associated with inoculated seedlings. In Experiment 1, T. koningii isolates 7a and 7c on autoclaved perlite substrate were mixed with pasteurized potting medium (Harris et a1., 1993a) at 20% vivo R. solani on wheat bran was mixed with potting medium at 0.35% v/v in a 7 mllayer. P. u. sporangiiferum on rice hulls was mixed with potting medium at 1.4% v/v throughout the punnet cell. In Experiment 2, the T. koningii isolates on autoclaved rice hulls were mixed throughout the potting medium at doses of 0.0175, 0.05, 0.14 or 0.42% v/v, and BNRI on rice hulls was mixed similarly at 0.42% vivo R. so[ani on wheat bran was mixed with potting medium at 3.0% v/v in a 7 mllayer. P. u. sporangiiferum on rice hulls was mixed with potting medium at 3.5% v/v throughout the punnet cell. All control treatments had 12 replicate punnets, and each BNRI treatment had 8 replicates. In Experiment 3, T. koningii isolates and BNRI were added as for experiment 2, except for additional doses of 7a and 7c at 1.2% v/v, and the dose of BNRI was increased to 1.0% vivo Sporania of P. u. sporangiiferum were produced using the method of Roberts and Lumsden (1990), and 2.25 ml of sporangial suspension was drenched onto the surface of the potting medium to deliver a dose of 260 sporangia cm-2. The control without pathogen was drenched similarly with sterile soil extract, and both controls and the BNRI treatment had 12 replicate punnets.
Table 1. Effect oftwo isolates of T. koningii (T. k.) and fungicides on survival and growth of Capsicum seedlings in pasteurized potting medium with either R. solani DIBI or P. u. sporangiiferum 2 (Experiment 1). Treatment
P. u. sporangiiferum
R. solani No. of plants survi ved/punneta
Dry weight of shoots, mg/punneta
No. of plants survived/punneta
Dry weight of shoots, mg/punneta
Control- (no pathogen): - uninoculated perlite - no perlite Control + pathogen
33.0 34.0 14.5
438 464 228
33.0 34.0 2.8
438 464 48
Pathogen + fungicide: - quintozene - propamocarb Pathogen + T. k. 7a Pathogen + T. k. 7c T. k. 7a alone T. k. 7c alone
32.8 ND 27.8 25.8 33.0 32.5
332 ND 297 325 394 395
NDb 24.0 18.5 23.5 33.0 32.5
ND 308 236 305 394 395
4.4 8.0
67 122
4.9 8.9
72 130
PLSD (P < 0.05) PLSD (P < 0.001)
a Each value is a mean from four punnets, each sown with 36 seeds. b ND = Not determined. 132
Microbiol. Res. 154 (1999) 2
Data for each variable were subjected to analysis of variance, and treatment means were compared to the controls by Fisher's protected least significant difference (PLSD) or standard error of the difference between two means (SED). The residuals from the analyses were examined and found to be approximately normally distributed and to have equal variance. No transformations were therefore used for the analyses of variance. Seedling survival data for doses of T. koningii isolates were subjected to a generalized linear regression, with binomial errors and a logistic link. This regression predicts A, which is a measure of seedling survival expressed as log (number survived/number died), in terms of dose of
T. koningii.
Results In Experiment 1, both isolates of T. koningii and the appropriate fungicide increased seedling survival in the presence of R. solani and P. u. sporangiiferum (P < 0.001), and the increases in shoot dry weights were significant at P = O.OS (Table 1). In the presence of R. solani, fewer seedlings survived (P < O.OS) with either T. koningii isolate than with quintozene. With Pythium, however, isolate 7c was as effective as propamocarb. Neither T. koningii isolate had a significant effect on seedling survival or shoot weight (P < O.OS) in the absence of pathogens. In Experiment 2, R. solani reduced the number of seedlings that survived from 33.4 in the control to lS.6 (S3% damped-off), but P. u. sporangiiferum only reduced it to 27.8 (17% damped-off) (Table 2). Regression analysis showed that both isolates of T. koningii gave linear negative dose responses in survival of seedlings with R. solani; the regression equation for T. koningii 7a was A = lS.6 - 1.S8 dose (SE = 0.73; t = -2.17), and for isolate 7c was A= IS.6- 2.43 dose (SE = 0.73; t= -3.36). There were no significant effects of either isolate of T. koningii against P. u. sporangiiferum (Table 2). Either isolate of T. koningii alone at the maximum dose did not have a significant effect on seedlings. BNRI suppressed R. solani more than T. koningii;with both seedling survival and shoot weight per punnet different (P < 0.01) from the positive and negative controls. Quintozene negated the effect of R. solani on seedling survival, but the shoot weight per punnet was intermediate and significantly different from both the positive and negative controls. BNRI also produced intermediate seedling survival with P. u. sporangiiferum, but had little effect on shoot weight per punnet. Propamocarb was moderately effective against Pythium, approximately halving the effects of the pathogen on seedling survival and shoot weight. In Experiment 3, the disease level was 4S% of seedlings damped-off (i.e., the difference between positive and negative controls) (Table 3). Both isolates of T. ko-
Table 2. Effect of two isolates of T. koningii (T. k.) at four doses, a binucleate Rhizoctonia isolate (BNRl) and fungicides on survival and growth of Capsicum seedlings in pasteurized potting medium with either R. solani DIBI or P. u. sporangiiferum 2 (Experiment 2). Treatment
No. of replicate punnetsa
Control- (no pathogen): 12 Control + R. solani 12 R. solani + quintozene 4 R. solani + T. k. 7a 0.0l75% 4 0.05% 4 0.14% 4 0.42% 4 R. solani + T. k. 7c 0.0l75% 4 0.05% 4 0.14% 4 0.42% 4 R. solani + BNRI 0.42% 8 Control + P. u. sporangiiferum 12 P. u. sporangiiferum + propamocarb 4 P. u. sporangiiferum + T. k. 7a 0.0175% 4 0.05% 4 0.14% 4 0.42% 4 P. u. sporangiiferum + T. k. 7c 0.0175% 4 0.05% 4 0.14% 4 0.42% 4 P. u. sporangiiferum + BNRI 0.42% 8 T. k. 7a alone, 0.42% 4 T. k. 7c alone, 0.42% 4 SED SED SED SED
4v4 4v12 8v12 12v12
No. plants Dry weight suvivedl of shoots, punnet mg/punnet 33.4 15.6 32.0
925 492 606
18.8 8.8 9.3 10.5
571 497 413 374
11.0 8.8 7.3 7.5 23.6
483 378 285 368 750
27.8
529
31.0
787
24.3 22.8 27.5 22.3
460 414 513 583
29.0 28.5 26.5 28.5
549 465 551 530
30.8 34.5 31.8
542 887 867
4.3 3.5 2.8 2.5
118 96 76 68
a Each punnet was sown with 36 seeds.
ningii gave dose responses in seedling survival with P. u. sporangiiferum. The regression equation for 7a was A = 20.8 + 1.18 dose (SE = 0.28; t = 4.27). The dose response of 7c was best described by a cubic regression. BNRI and propamocarb also suppressed disease (Table 3). Neither isolate of T. koningii alone at the maximum dose had a significant effect on seedlings. Re-isolations of the inoculated fungal genera from roots of randomly selected seedlings confirmed that the inoculations were successful. Microbiol. Res. 154 (1999) 2
133
Table 3. Effect of two isolates of T. koningii (T. k.) at five doses, a binucleate Rhizoctonia isolate (BNRl) and a fungicide on survival and growth of Capsicum seedlings in pasteurized potting medium with P. u. sporangiiferum 2 (Experiment 3). No. of replicate punnets a
Treatment
No. plants Dry weight suvivedJ of shoots, mg/punnet punnet 30.3
1305
16.7
809
4
25.8
932
4 4 4 4 4
21.5 26.5 28.8 27.8 33.3
978 1429 1287 1371 1607
4 4 4 4 4
19.3 18.5 26.8 9.8 13.3
852 981 1387 491 649
P. u. sporangiiferum + BNR11.0% T. k. 7a alone, 1.2 % T. k. 7c alone, 1.2 %
12 4 4
25.7 31.5 33.8
1342 1610 1648
SED SED SED
4v4 4v12 l2v12
3.0 2.5 1.7
149 122 86
Control- (no pathogen): 12 Control + P. u. 12 sporangiiferum P. u. sporangiiferum + propamocarb P. u. sporangiiferum + T. k. 7a 0.0175%
0.05% 0.14% 0.42% 1.2% P. u. sporangiiferum + T. k. 7c 0.0175%
0.05% 0.14% 0.42% 1.2%
situations is likely to vary since their biology, and probably their modes of action, are very different. For BNR1, mycoparasitism (Siwek et at., 1997 a), antibiosis (Siwek et at., 1997b), competitive saprophytic ability (Siwek, 1996) and seedling protection through colonization and competition (Harris et at., 1997) may all be important in bioncontrol. However, antibiosis appears to be the major biocontrol mechanism for T. koningii (Simon et at., 1988) and Gliocladium (Trichoderma) virens (Roberts and Lumsden, 1990; Harris and Lumsden, 1997) and mycoparasitism is unimportant, at least against R. solani AG 4 (Howell, 1987). Further research is needed to determine whether either of the T. koningii isolates could be used alone or in combination with other biocontrol microorganisms for control of phytopathogenic soil-borne fungi on bedding plants in some situations. T. koningii may be useful in commercial situations where BNR1 is not fully effective, such as where other plant pathogens are present (Harris and Nelson, 1999).
Acknowledgements I thank Incitec Ltd, Brisbane, Queensland for partial financial support; T. E. Terrace and P. G. Adkins for excellent technical assistance; the late A. Simon for supplying cultures of T. koningii; R. L. Correll of CSIRO Mathematical and Information Sciences for help with experimental designs and statistical analyses; and Falg Nurseries Pty Ltd, Uraidla, South Australia for supplying potting medium and punnets.
a Each punnet was sown with 36 seeds.
References Discussion Both isolates of T. koningii increased survival of Capsicum seedlings in the presence of R. solani AG 4 or P. u. sporangiiferum in Experiments 1 and 3, although isolate 7c was not effective at all doses against P. u. sporangiiferum. In Experiment 2, the reason for the lack of damping-off control by either T. koningii isolate with R. solani is not known. The negative dose responses cannot be due to direct effects of the T. koningii on the seedlings because neither T. koningii isolate at maximum dose in the absence of pathogens had any detrimental effect on seedlings in any experiment. Although T. koningii 7a was as effective as BNR1 against P. u. sporangiiferum in Experiment 3, neither T. koningii isolate suppressed the two damping-off pathogens as consistently as BNRI. BNRI is an effective biocontrol agent under a wide range of conditions (Harris and Adkins, 1999). However, the comparative biocontrol efficacy of BNR1 and T. koningii in different 134
Microbiol. Res. 154 (1999) 2
Cline, M. N., Chastagner, G. A., Aragaki, M., Baker, R., Daughtrey, M. L., Lawson, R. H., MacDonald, 1. D., Tammen, 1. E, Worf, G. L. (1988): Current and future research directions of ornamental pathology. Plant Dis. 72,926-934. Fravel, D. R, Connick, W. 1. Jr., Lewis, 1. A. (1996): Formulation of microorganisms to control plant diseases. In: Formulation of Microbial Biopesticides, Beneficial Microorganisms and Nematodes (Ed: Burges, H. D.). Chapman and Hall, London. Harris, A. R (1995): Damping-off survey supports research. Australian Nursery Magazine 19(2),12-13. Harris, A. R (1999): Plant growth promotion by binucleate Rhizoctonia and bacterial isolates in mono xenic cultures. Microbiol. Res. 154, (in press). Harris, A. R, Adkins, P. G. (1999): Versatility of fungal and bacterial isolates for biological control of damping-off disease caused by Rhizoctonia solani and Pythium spp. Biological Control 15, 10-18. Harris, A. R, Lumsden, R D. (1997): Interactions of Gliocladium virens with Rhizoctonia solani and Pythium ultimum in non-sterile potting medium. Biocontrol Sci. Technol. 7, 37-47.
Harris, A. R., Nelson, S. (1999): Progress towards integrated control of damping-off disease. Microbiol. Res. 154, (in press). Harris, A. R., Schisler, D. A., Neate, S. M. (l993a): Culture of Rhizoctonia solani and binucleate Rhizoctonia spp. on organic substrates for inoculation of seedlings in containers. Soil BioI. Biochem. 25, 337-34l. Harris, A. R., Schisler, D. A., Ryder, M. H. (1993b): Binucleate Rhizoctonia isolates control damping-off caused by Pythium ultimum var. sporangiiferum, and promote growth in Capsicum and Celosia seedlings in pasteurized potting medium. Soil BioI. Biochem. 25, 909-914. Harris, A. R, Schisler, D. A., Correll, R. L., Ryder, M. H. (1994 a): Soil bacteria selected for suppression of Rhizoctonia solani, and growth promotion in bedding plants. Soil BioI. Biochem. 26,1249-1255. Harris, A. R, Schisler, D. A., Neate, S. M., Ryder, M. H. (1994 b): Suppression of damping-off caused by Rhizoctonia solani, and growth promotion in bedding plants by binucleate Rhizoctonia spp. Soil BioI. Biochem. 26, 263-268. Harris, A. R, Schisler, D. A., Ryder, M. H. Adkins, P. G. (1994c): Bacteria suppress damping-off caused by Pythium ultimum var. sporangiiferum, and promote growth, in bedding plants. Soil BioI. Biochem. 26, 1431-1437. Harris, A. R, Siwek, K., Wiseman, B. M. (1997): Interactions between damping-off fungi, antagonists and Capsicum seedlings. Appl. Soil Ecol. 6, 251-263. Howell, C. R. (1987): Relevance of mycoparasitism in the biological control of Rhizoctonia solani by Gliocladium virens. Phytopathology 77, 992-994. Papavizas, G. C. (1985): Trichoderma and Gliocladium:
biology, ecology, and potential for biocontrol. Annu. Rev. Phytopathol. 23, 23-54. Roberts, D. P., Lumsden, RD. (1990): Effect of extracellular metabolites from Gliocladium virens on germination of sporangia and mycelial growth of Pythium ultimum. Phytopathology 80, 461-465. Simon, A. (1989): Biological control of take-all of wheat by Trichoderma koningii under controlled environmental conditions. Soil BioI. Biochem. 21, 323-326. Simon, A., Dunlop, R W:, Ghisalberti, E. L., Sivasithamparam, K. (1988): Trichoderma koningii produces a pyrone compound with antibiotic properties. Soil BioI. Biochem. 20,263-264. Siwek, K. (1996): Mechanisms of biological control of the damping-off fungus, Pythium ultimum, by binucleate Rhizoctonia. Ph.D. thesis, Univ. Adelaide, Australia. Siwek, K., Harris, A. R., Scott, E. S. (1997 a): Mycoparasitism of Pythium ultimum by antagonistic binucleate Rhizoctonia isolates in agar media and on Capsicum seeds. 1. Phytopathoi. 145,417-423. Siwek, K., Scott, E. S., Harris, A. R (1997b): Role of antibiosis in biological control of Pythium ultimum by binucleate Rhizoctonia. Abstract 11 th Australian Plant Pathology Society Conference, Perth, Australia. 29 September - 2 October, 1997. Stephens, C. T., Powell, C. C. (1982): Pythium species causing damping-off of seedling bedding plants in Ohio greenhouses. Plant Dis. 66, 731-733. Stephens, C. T., Herr, L. 1., Schmitthenner, A. F. (1982): Characterization of Rhizoctonia isolates associated with damping-off of bedding plants. Plant Dis. 66, 700-703.
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Urban & Fischer Verlag
Biocontrol of Rhizoctonia solani and Pythium ultimum on Capsicum by Trichoderma koningii in potting medium A. R. Harris
CSIRO Division of Soils, and Cooperative Research Centre for Soil and Land Management, Private Bag No.2, Glen Osmond, S.A. 5064, Australia Accepted : June 17, 1999
Abstract Two isolates of Trichoderma koningii were evaluated for efficacy in control of damping-off diseases in seedlings of Capsicum annuum grown in pasteurized potting medium in a glasshouse. A selected isolate of binucleate Rhizoctonia and two fungicides were also included as standards for control of Rhizoctonia solani and Pythium ultimum var. sporangiiferum. Both isolates of T. koningii reduced seedling death caused by R. solani in one of two experiments, and by P. u. sporangiiferum in two of three experiments. Neither isolate of T. koningii suppressed damping-off caused by either pathogen as consistently as the binucleate Rhizoctonia or fungicides. The implications of these results for commercial disease management are discussed. Key words: Trichoderma koningii - binucleate Rhizoctonia Pythium ultimum var. sporangiiferum - Rhizoctonia solani Capsicum annuum - biological control- damping-off - potting medium - fungicides
(Stephens et al., 1982). In previous papers, we have reported on the efficacy of selected isolates of soil bacteria and binucleate Rhizoctonia, including BNR1, for suppression of these pathogenic fungi (Harris et al., 1993 b, 1994 a, b, c; Harris and Adkins, 1999), and plant growth promotion (Harris, 1999). Trichoderma spp. are important biological control agents (Papavizas, 1985), and several products that contain Trichoderma spp. are sold to control a variety of plant pathogens (Fravel et al., 1996). Isolates of T. koningii Oudem. have been shown to be antagonistic to soil-borne pathogenic fungi (Simon, 1989), including R. solani AG 8, Pythium middletonii and Phytophthora cinnamomi (Simon et ai., 1988). The aim of this research was to determine whether T. koningii isolates 7a and 7c have potential for control of damping-off diseases in bedding plants, and to compare their efficacy with one of our selected binucleate Rhizoctonia isolates, BNRl.
Introduction
Materials and methods
The nursery industry needs an economical, environmentally safe biological product to control seedling damping-off diseases caused by Pythium spp. and Rhizoctonia solani KUhn (teleomorph = Thanatephorus cucumeris (Frank) Donk) (Stephens and Powell, 1982; Cline et al., 1988; Harris, 1995). In the Ohio bedding plant industry, damping-off attributed to Rhizoctonia is caused primarily by R. soiani anastomosis group (AG) 4
T. koningii isolates 7a and 7c were isolated originally from wheat soil in Western Australia that was suppressive to Gaeumannomyces graminis var. tritici (Simon, 1989). Pythium ultimum var. sporangiiferum Drechsler isolate 2, R. solani isolate DIBI (AG 4) and binucleate Rhizoctonia isolate BNRI (AG-K) were isolated from plant nurseries around Adelaide, South Australia (Harris et al., 1993 a, 1993 b). These fungi were cultured on sterilized organic substrates and added to pasteurized potting medium (pH(OOlMCaC12) = 5.6) in six-celled plastic punnets (seedling trays) (Harris et at., 1993a). R. solani on wheat bran was mixed with potting medium, and 7 cm3 of this mix was added to the bottom of
Corresponding author: A. R. Harris, Present address: Australian Quarantine and Inspection Service, GPO Box 858, Canberra, A.c.T. 2601, Australia. (E-mail: [email protected]) 0944-5013/99/154/02-131
$12.00/0
Microbiol. Res. 154 (1999) 2
131
each punnet cell. Punnets then were filled with either uninfested potting medium or potting medium infested with BNRI and/or P. u. sporangiiferum. BNRI and P. u. sporangiiferum were cultured on pulverized rice hulls (Harris et a1., 1993 a). Fungal doses are given below. Six seeds of Capsicum annuum L. 'Green Giant' (capsicum, syn. bell pepper) were sown near the top of the potting medium in each punnet cell, and covered with 5 -1 0 mm (approx. 7 cm3) of sterilized, washed coarse sand. The biological control efficacy of the selected fungal antagonists was compared with the fungicides quintozene and propamocarb, which are registered in Australia for control of Rhizoctonia and Pythium respectively in some seedlings. Quintozene (PCNB) (Terraclor, Uniroyal Australia) was suspended in deionized water at 5 g 1-1, and propamocarb (Previcur, Schering AG) at 1.78 mIl-I. 2.25 ml of each suspension was pipetted onto the soil surface in each punnet cell to give applied doses of quintozene of 8.4 mg active ingedient (a.i.) per punnet cell and propamocarb of 2.4 mg a.i. per punnet cell, which approximate the recommended commercial rates. Punnets were arranged in randomized complete block designs with four blocks in a glasshouse at 25°C (± 5°C), and watered twice weekly with a nutrient solution (Harris et az', 1993b). When most seedlings had four true leaves and damping-off had ceased (3-4 weeks), the surviving and collapsed seedlings were counted separately, the standing seedlings were excised at soil level, and the tops were dried at 60°C and then weighed. Root samples from randomly selected punnet cells containing each fungus were washed, plated onto water
agar (Oxoid No.3, Unipath Ltd., Basingstoke, Hampshire, England) or 114-strength potato dextrose agar (Difco Laboratories, Detroit, Mich.), and incubated at 25°C to confirm that the added fungal genera were associated with inoculated seedlings. In Experiment 1, T. koningii isolates 7a and 7c on autoclaved perlite substrate were mixed with pasteurized potting medium (Harris et a1., 1993a) at 20% vivo R. solani on wheat bran was mixed with potting medium at 0.35% v/v in a 7 mllayer. P. u. sporangiiferum on rice hulls was mixed with potting medium at 1.4% v/v throughout the punnet cell. In Experiment 2, the T. koningii isolates on autoclaved rice hulls were mixed throughout the potting medium at doses of 0.0175, 0.05, 0.14 or 0.42% v/v, and BNRI on rice hulls was mixed similarly at 0.42% vivo R. so[ani on wheat bran was mixed with potting medium at 3.0% v/v in a 7 mllayer. P. u. sporangiiferum on rice hulls was mixed with potting medium at 3.5% v/v throughout the punnet cell. All control treatments had 12 replicate punnets, and each BNRI treatment had 8 replicates. In Experiment 3, T. koningii isolates and BNRI were added as for experiment 2, except for additional doses of 7a and 7c at 1.2% v/v, and the dose of BNRI was increased to 1.0% vivo Sporania of P. u. sporangiiferum were produced using the method of Roberts and Lumsden (1990), and 2.25 ml of sporangial suspension was drenched onto the surface of the potting medium to deliver a dose of 260 sporangia cm-2. The control without pathogen was drenched similarly with sterile soil extract, and both controls and the BNRI treatment had 12 replicate punnets.
Table 1. Effect oftwo isolates of T. koningii (T. k.) and fungicides on survival and growth of Capsicum seedlings in pasteurized potting medium with either R. solani DIBI or P. u. sporangiiferum 2 (Experiment 1). Treatment
P. u. sporangiiferum
R. solani No. of plants survi ved/punneta
Dry weight of shoots, mg/punneta
No. of plants survived/punneta
Dry weight of shoots, mg/punneta
Control- (no pathogen): - uninoculated perlite - no perlite Control + pathogen
33.0 34.0 14.5
438 464 228
33.0 34.0 2.8
438 464 48
Pathogen + fungicide: - quintozene - propamocarb Pathogen + T. k. 7a Pathogen + T. k. 7c T. k. 7a alone T. k. 7c alone
32.8 ND 27.8 25.8 33.0 32.5
332 ND 297 325 394 395
NDb 24.0 18.5 23.5 33.0 32.5
ND 308 236 305 394 395
4.4 8.0
67 122
4.9 8.9
72 130
PLSD (P < 0.05) PLSD (P < 0.001)
a Each value is a mean from four punnets, each sown with 36 seeds. b ND = Not determined. 132
Microbiol. Res. 154 (1999) 2
Data for each variable were subjected to analysis of variance, and treatment means were compared to the controls by Fisher's protected least significant difference (PLSD) or standard error of the difference between two means (SED). The residuals from the analyses were examined and found to be approximately normally distributed and to have equal variance. No transformations were therefore used for the analyses of variance. Seedling survival data for doses of T. koningii isolates were subjected to a generalized linear regression, with binomial errors and a logistic link. This regression predicts A, which is a measure of seedling survival expressed as log (number survived/number died), in terms of dose of
T. koningii.
Results In Experiment 1, both isolates of T. koningii and the appropriate fungicide increased seedling survival in the presence of R. solani and P. u. sporangiiferum (P < 0.001), and the increases in shoot dry weights were significant at P = O.OS (Table 1). In the presence of R. solani, fewer seedlings survived (P < O.OS) with either T. koningii isolate than with quintozene. With Pythium, however, isolate 7c was as effective as propamocarb. Neither T. koningii isolate had a significant effect on seedling survival or shoot weight (P < O.OS) in the absence of pathogens. In Experiment 2, R. solani reduced the number of seedlings that survived from 33.4 in the control to lS.6 (S3% damped-off), but P. u. sporangiiferum only reduced it to 27.8 (17% damped-off) (Table 2). Regression analysis showed that both isolates of T. koningii gave linear negative dose responses in survival of seedlings with R. solani; the regression equation for T. koningii 7a was A = lS.6 - 1.S8 dose (SE = 0.73; t = -2.17), and for isolate 7c was A= IS.6- 2.43 dose (SE = 0.73; t= -3.36). There were no significant effects of either isolate of T. koningii against P. u. sporangiiferum (Table 2). Either isolate of T. koningii alone at the maximum dose did not have a significant effect on seedlings. BNRI suppressed R. solani more than T. koningii;with both seedling survival and shoot weight per punnet different (P < 0.01) from the positive and negative controls. Quintozene negated the effect of R. solani on seedling survival, but the shoot weight per punnet was intermediate and significantly different from both the positive and negative controls. BNRI also produced intermediate seedling survival with P. u. sporangiiferum, but had little effect on shoot weight per punnet. Propamocarb was moderately effective against Pythium, approximately halving the effects of the pathogen on seedling survival and shoot weight. In Experiment 3, the disease level was 4S% of seedlings damped-off (i.e., the difference between positive and negative controls) (Table 3). Both isolates of T. ko-
Table 2. Effect of two isolates of T. koningii (T. k.) at four doses, a binucleate Rhizoctonia isolate (BNRl) and fungicides on survival and growth of Capsicum seedlings in pasteurized potting medium with either R. solani DIBI or P. u. sporangiiferum 2 (Experiment 2). Treatment
No. of replicate punnetsa
Control- (no pathogen): 12 Control + R. solani 12 R. solani + quintozene 4 R. solani + T. k. 7a 0.0l75% 4 0.05% 4 0.14% 4 0.42% 4 R. solani + T. k. 7c 0.0l75% 4 0.05% 4 0.14% 4 0.42% 4 R. solani + BNRI 0.42% 8 Control + P. u. sporangiiferum 12 P. u. sporangiiferum + propamocarb 4 P. u. sporangiiferum + T. k. 7a 0.0175% 4 0.05% 4 0.14% 4 0.42% 4 P. u. sporangiiferum + T. k. 7c 0.0175% 4 0.05% 4 0.14% 4 0.42% 4 P. u. sporangiiferum + BNRI 0.42% 8 T. k. 7a alone, 0.42% 4 T. k. 7c alone, 0.42% 4 SED SED SED SED
4v4 4v12 8v12 12v12
No. plants Dry weight suvivedl of shoots, punnet mg/punnet 33.4 15.6 32.0
925 492 606
18.8 8.8 9.3 10.5
571 497 413 374
11.0 8.8 7.3 7.5 23.6
483 378 285 368 750
27.8
529
31.0
787
24.3 22.8 27.5 22.3
460 414 513 583
29.0 28.5 26.5 28.5
549 465 551 530
30.8 34.5 31.8
542 887 867
4.3 3.5 2.8 2.5
118 96 76 68
a Each punnet was sown with 36 seeds.
ningii gave dose responses in seedling survival with P. u. sporangiiferum. The regression equation for 7a was A = 20.8 + 1.18 dose (SE = 0.28; t = 4.27). The dose response of 7c was best described by a cubic regression. BNRI and propamocarb also suppressed disease (Table 3). Neither isolate of T. koningii alone at the maximum dose had a significant effect on seedlings. Re-isolations of the inoculated fungal genera from roots of randomly selected seedlings confirmed that the inoculations were successful. Microbiol. Res. 154 (1999) 2
133
Table 3. Effect of two isolates of T. koningii (T. k.) at five doses, a binucleate Rhizoctonia isolate (BNRl) and a fungicide on survival and growth of Capsicum seedlings in pasteurized potting medium with P. u. sporangiiferum 2 (Experiment 3). No. of replicate punnets a
Treatment
No. plants Dry weight suvivedJ of shoots, mg/punnet punnet 30.3
1305
16.7
809
4
25.8
932
4 4 4 4 4
21.5 26.5 28.8 27.8 33.3
978 1429 1287 1371 1607
4 4 4 4 4
19.3 18.5 26.8 9.8 13.3
852 981 1387 491 649
P. u. sporangiiferum + BNR11.0% T. k. 7a alone, 1.2 % T. k. 7c alone, 1.2 %
12 4 4
25.7 31.5 33.8
1342 1610 1648
SED SED SED
4v4 4v12 l2v12
3.0 2.5 1.7
149 122 86
Control- (no pathogen): 12 Control + P. u. 12 sporangiiferum P. u. sporangiiferum + propamocarb P. u. sporangiiferum + T. k. 7a 0.0175%
0.05% 0.14% 0.42% 1.2% P. u. sporangiiferum + T. k. 7c 0.0175%
0.05% 0.14% 0.42% 1.2%
situations is likely to vary since their biology, and probably their modes of action, are very different. For BNR1, mycoparasitism (Siwek et at., 1997 a), antibiosis (Siwek et at., 1997b), competitive saprophytic ability (Siwek, 1996) and seedling protection through colonization and competition (Harris et at., 1997) may all be important in bioncontrol. However, antibiosis appears to be the major biocontrol mechanism for T. koningii (Simon et at., 1988) and Gliocladium (Trichoderma) virens (Roberts and Lumsden, 1990; Harris and Lumsden, 1997) and mycoparasitism is unimportant, at least against R. solani AG 4 (Howell, 1987). Further research is needed to determine whether either of the T. koningii isolates could be used alone or in combination with other biocontrol microorganisms for control of phytopathogenic soil-borne fungi on bedding plants in some situations. T. koningii may be useful in commercial situations where BNR1 is not fully effective, such as where other plant pathogens are present (Harris and Nelson, 1999).
Acknowledgements I thank Incitec Ltd, Brisbane, Queensland for partial financial support; T. E. Terrace and P. G. Adkins for excellent technical assistance; the late A. Simon for supplying cultures of T. koningii; R. L. Correll of CSIRO Mathematical and Information Sciences for help with experimental designs and statistical analyses; and Falg Nurseries Pty Ltd, Uraidla, South Australia for supplying potting medium and punnets.
a Each punnet was sown with 36 seeds.
References Discussion Both isolates of T. koningii increased survival of Capsicum seedlings in the presence of R. solani AG 4 or P. u. sporangiiferum in Experiments 1 and 3, although isolate 7c was not effective at all doses against P. u. sporangiiferum. In Experiment 2, the reason for the lack of damping-off control by either T. koningii isolate with R. solani is not known. The negative dose responses cannot be due to direct effects of the T. koningii on the seedlings because neither T. koningii isolate at maximum dose in the absence of pathogens had any detrimental effect on seedlings in any experiment. Although T. koningii 7a was as effective as BNR1 against P. u. sporangiiferum in Experiment 3, neither T. koningii isolate suppressed the two damping-off pathogens as consistently as BNRI. BNRI is an effective biocontrol agent under a wide range of conditions (Harris and Adkins, 1999). However, the comparative biocontrol efficacy of BNR1 and T. koningii in different 134
Microbiol. Res. 154 (1999) 2
Cline, M. N., Chastagner, G. A., Aragaki, M., Baker, R., Daughtrey, M. L., Lawson, R. H., MacDonald, 1. D., Tammen, 1. E, Worf, G. L. (1988): Current and future research directions of ornamental pathology. Plant Dis. 72,926-934. Fravel, D. R, Connick, W. 1. Jr., Lewis, 1. A. (1996): Formulation of microorganisms to control plant diseases. In: Formulation of Microbial Biopesticides, Beneficial Microorganisms and Nematodes (Ed: Burges, H. D.). Chapman and Hall, London. Harris, A. R (1995): Damping-off survey supports research. Australian Nursery Magazine 19(2),12-13. Harris, A. R (1999): Plant growth promotion by binucleate Rhizoctonia and bacterial isolates in mono xenic cultures. Microbiol. Res. 154, (in press). Harris, A. R, Adkins, P. G. (1999): Versatility of fungal and bacterial isolates for biological control of damping-off disease caused by Rhizoctonia solani and Pythium spp. Biological Control 15, 10-18. Harris, A. R, Lumsden, R D. (1997): Interactions of Gliocladium virens with Rhizoctonia solani and Pythium ultimum in non-sterile potting medium. Biocontrol Sci. Technol. 7, 37-47.
Harris, A. R., Nelson, S. (1999): Progress towards integrated control of damping-off disease. Microbiol. Res. 154, (in press). Harris, A. R., Schisler, D. A., Neate, S. M. (l993a): Culture of Rhizoctonia solani and binucleate Rhizoctonia spp. on organic substrates for inoculation of seedlings in containers. Soil BioI. Biochem. 25, 337-34l. Harris, A. R., Schisler, D. A., Ryder, M. H. (1993b): Binucleate Rhizoctonia isolates control damping-off caused by Pythium ultimum var. sporangiiferum, and promote growth in Capsicum and Celosia seedlings in pasteurized potting medium. Soil BioI. Biochem. 25, 909-914. Harris, A. R, Schisler, D. A., Correll, R. L., Ryder, M. H. (1994 a): Soil bacteria selected for suppression of Rhizoctonia solani, and growth promotion in bedding plants. Soil BioI. Biochem. 26,1249-1255. Harris, A. R, Schisler, D. A., Neate, S. M., Ryder, M. H. (1994 b): Suppression of damping-off caused by Rhizoctonia solani, and growth promotion in bedding plants by binucleate Rhizoctonia spp. Soil BioI. Biochem. 26, 263-268. Harris, A. R, Schisler, D. A., Ryder, M. H. Adkins, P. G. (1994c): Bacteria suppress damping-off caused by Pythium ultimum var. sporangiiferum, and promote growth, in bedding plants. Soil BioI. Biochem. 26, 1431-1437. Harris, A. R, Siwek, K., Wiseman, B. M. (1997): Interactions between damping-off fungi, antagonists and Capsicum seedlings. Appl. Soil Ecol. 6, 251-263. Howell, C. R. (1987): Relevance of mycoparasitism in the biological control of Rhizoctonia solani by Gliocladium virens. Phytopathology 77, 992-994. Papavizas, G. C. (1985): Trichoderma and Gliocladium:
biology, ecology, and potential for biocontrol. Annu. Rev. Phytopathol. 23, 23-54. Roberts, D. P., Lumsden, RD. (1990): Effect of extracellular metabolites from Gliocladium virens on germination of sporangia and mycelial growth of Pythium ultimum. Phytopathology 80, 461-465. Simon, A. (1989): Biological control of take-all of wheat by Trichoderma koningii under controlled environmental conditions. Soil BioI. Biochem. 21, 323-326. Simon, A., Dunlop, R W:, Ghisalberti, E. L., Sivasithamparam, K. (1988): Trichoderma koningii produces a pyrone compound with antibiotic properties. Soil BioI. Biochem. 20,263-264. Siwek, K. (1996): Mechanisms of biological control of the damping-off fungus, Pythium ultimum, by binucleate Rhizoctonia. Ph.D. thesis, Univ. Adelaide, Australia. Siwek, K., Harris, A. R., Scott, E. S. (1997 a): Mycoparasitism of Pythium ultimum by antagonistic binucleate Rhizoctonia isolates in agar media and on Capsicum seeds. 1. Phytopathoi. 145,417-423. Siwek, K., Scott, E. S., Harris, A. R (1997b): Role of antibiosis in biological control of Pythium ultimum by binucleate Rhizoctonia. Abstract 11 th Australian Plant Pathology Society Conference, Perth, Australia. 29 September - 2 October, 1997. Stephens, C. T., Powell, C. C. (1982): Pythium species causing damping-off of seedling bedding plants in Ohio greenhouses. Plant Dis. 66, 731-733. Stephens, C. T., Herr, L. 1., Schmitthenner, A. F. (1982): Characterization of Rhizoctonia isolates associated with damping-off of bedding plants. Plant Dis. 66, 700-703.
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