RHIZOCTONIA AND PYTHIUM UNDER CONTROLLED GREENHOUSE CONDITIONS
4.2 MATERIALS AND METHODS .1 Source of materials
Grain sorghum (cv. PAN8446) obtained from Pannar3 Seeds and Eragrostis tef (cv. SA Brown) were obtained from McDonalds4 Seeds. Bacillus spp. H44 and BSI were originally isolated from a soil collected from a sorghum field at Cedara, Kwazulu-Natal, South Africa.
Isolate B81 was provided by the Discipline of Plant Pathology', University of Natal.
Trichoderma harzianum (Eco-T) and Trichoderma spp. SY3 and SY4 were supplied by Plant
IK.S. Yobo, Discipline of Plant Pathology, School of Applied Environmental Sciences, University of Natal, Private Bag X01, Scottsville, 3209, South Africa
2B. Kubheka, Discipline of Plant Pathology, School of Applied Environmental Sciences, University of Natal, PrivateBag X01, Scottsville, 3209, South Africa
3Pannar Seeds (Pty), P.O.Box 19, Greytown 3250, South Africa
4McDonaldsSeeds, 61 Boshoff Street, P.O.Box238, Pieterrnaritzburg, South Africa
5Discipline of Plant Pathology, School of Applied Environmental Sciences, University of Natal, Private Bag X01, Scottsville, 3209, South Africa
Health Products6 in formulation forms consisting of 108 colony forming units per gram (c.f.u g-!). Pythium sp. and R. solani were obtained from C.C. Clare. Except for the Trichoderma spp. that were in a formulation form, all microbes were maintained on agar slants and subcultured on V-8 agar when needed.
4.2.2 Preparation of antagonistic bacteria and fungi
All isolates of Bacillus were grown on Potato Dextose Agar (PDA) for two days and inoculated into conical flasks (250ml) containing lOOml of Tryptone Soy Broth (TSB). The flasks were placed in a rotary shaker (120rpm) for 48 hat 23
±
2°C. The resultant suspension was centrifuged for 15min at 9,000rpm. The broth was decanted and the pellet of cells resuspended in sterilized distilled water. Counting0 fb acterial c .f.u. was d one after 24h 0f incubation on Tryptone Soy Agar (TSA) at 25-28°C. The concentrations of Bacillus and Trichoderma were adjusted to l09and l05c.f.u. mr! of sterile distilled water, respectively.4.2.3 Biological treatments
• Seed coating
A microbial-sticker mixture was prepared by rmxmg 2g of CMC with 98ml microbial suspension of the specified concentrationsinto a conical flask (250ml). Mixtures of microbial- sticker were dissolved by stirring vigorously and allowed to stand for lh to obtain a uniform mixture. Then 109 of seeds were immersed into lOml of the mixture for l2-l5min. All treated seeds were placed on filter paper and air-dried overnight. Coated seeds were stored at 4°C and planted no longer than five days after treatment (Mao et al., 1998a). Speedling" 24 trays containing coated seeds were left in the planting room overnight and only watered the following day to avoid removal of microbes before their establishment. At the first sign of germination, Speedling'" 24 trays were transferred to a greenhouse opertating at temperature of 26-28°C and relative humuidty of75-85%.
6Dr. M.Morris, PlantHealth Products (Pty) Ltd; P.G.Box 207, Nottingham Road, South Africa
7C. Clark, Discipline of Plant Pathology, School of Applied Environmental Sciences, University of Natal, Private Bag XOl, Scottsville, 3209, South Africa
• Drenching
Untreated seeds of sorghum and tef were planted into Speedling'" 24 trays. Before seeds were covered, 1ml of Trichoderma (105 c.f.u. ml") or Bacillus (109c.f.u. mrl) suspension was drenched directly onto seeds in their respective trays. All Speedling'" 24 trays were left in .the planting room overnight, and only watered the following day to allow establishment of microbes on the target seed. At the first sign of germination, Speedling'" 24 trays were transferred to a greenhouse operating at temperatures of 26-28 QC and relative humidity 75- 85%.
4.2.4 Other treatments
Control treatments consisted of clean (seeds without CMC) or untreated (seeds coated only with CMC) seeds were planted in infested (with pathogen) or non-infested (without pathogen) soils. For the chemical treatments, untreated seeds were planted into Speedling'" 24 trays and covered with composted pine bark and drenched with 3ml of Benlate®8 at 1.0g I-I and Previcur®9 at 1.2ml
r'
during planting to control R. solani and Pythium sp., respectively.Sprayed trays were left in the planting room overnight and watered the next day to avoid immediate leaching of chemicals. At the first sign of germination, the Speedling" 24 trays were transferred to the greenhouse.
4.2.5 Artificial inoculation of the media with pathogens
Initially, a thin layer of composted pine bark was placed in Speedling'" 24 trays. A 4mm square agar plug, containing the pathogen of a five day-old culture was placed on each cell above the composted pine bark and slightly covered with additional composted pine bark. The media was then gently watered. One seed was placed on the top of the pine bark before treatments were applied, resulting in 24 seedsper tray and 96 seeds per treatment.
8El du Ponte de Nemours and Co., Wilmington, Delaware. 19898, USA
9AgroEvo South Africa (Pty), P.O.Box 6065, Halfway House 1685, South Africa
4.2.6 Variables of seedlings
Assessment of pre- and post-emergence damping-off caused by a Pythium sp. and R. so/ani was conducted by counting the number of emerged seedlings two weeks after planting and the percentage of seedlings that survived four (for tef) and six (for sorghum) weeks of growth. All surviving seedlings in each tray were cut at their base, oven dried for two days at 70DC and then weighed.
4.2.7 Statistical analysis
Each treatment had four replicates, each plot having 24 seedlings hence, 96 seedlings per treatment. Treatments were arranged in a randomized complete block design. The experiment was conducted twice. Data obtained from the two trials were analysed three times using Genstat'" Statistical Analysis Software (Genstat, 2002). Firstly, data of the two trials were analyzed separately to determine Analysis of Variance (ANOVA), to make contrasts between infected and uninfected controls, to determine the effect of the sticker on seedling emergence and to separated treatment means using Fisher's Protected LSD. Secondly, data0 f t he two trials were combined and analysed together to determine the effect of season on treatments.
Thirdly, data of the two trials containing only results of BCAs were combined together and analysed to determine interactive effects between season, application method and BCAs and to compare the two application methods. In each analysis, transformation of data using square root transformations was performed where CV of the original data was >20% and the statistical results generated from the transformed data were used to meet the objectives.