HELMINTHOSPORIUM LEAF SPOT ON THE PANICOID GRASS USING AMENDED BIOCONTROL AGENTS
4.2 MATERIALS AND METHODS
4.2.3 FIELD TRIALS
Prepared formulations of antagonistic Bacillus and Trichoderma strains were obtained for in vivo testing. For the 2000 trial, Bacillus strains were applied as a prepared liquid formulation marketed as BIOSTART®2000 (designated as BIOSTART), comprising 4 partsB.laterosporusLaubach; 4 partsB.chitinosporusLA6A and 2 partsB.licheniformis Weigmann. Trichoderma was applied as ROOTSHIELD® (designated as ROOTSHIELD), comprising T. harzianum. Both BIOSTART and ROOTSHIELD were obtained from Microbial Solutions1.
BIOSTART and ROOTSHIELD were not available for the 2002 trial. Instead, Bacillus subtilisB69 in talcum powder (designated Bacillus B69) and Trichoderma harzianum kd in shredded wheat (designated Trichoderma kd), were used. Trichoderma kd and Bacillus B69 were obtained from Plant Health Products
cc",
The product based on Bacillus B69 is still undergoing performance tests before commercial release.2
Microbial Solutions (Pty) Ltd.,p.a.Box 103, Kya Sand, 2163, South Africa. Tel: (+27) 11 462-2408 Plant Health Products cc., p.a.Box 207, Nottingham Road, 3280, South Africa. Tel: (+27) 33263 6130
The antagonistic potential of these two microorganisms were measured against the fungicide PUNCH XTRA® (designated PUNCH XTRA), registered for various leaf spot diseases on a number of hosts (Nel et al., 1999). A water control (zero microbial treatment) was also included for comparison.
Within the treatment types, dosage rates were also assessed for the level disease control. Treatments were applied at zero (Le. control), half and the full manufacturer's recommended dose.
Field trials were conducted at Cedara(Department of Agriculture and Environmental Affairs) in the KwaZulu-Natal Midlands, 32km inland from Pietermaritzburg, South Africa.
4.2.3.1 2000 trial Trial site
A pure stand ofP. clandestinum with heavy infestations of Bipolaris was chosen as the trial area.
Trial design
The trial design was that of a randomised complete block design, with nine treatments and five replicates, laid out into 45, 1m2 plots. A border of O.5m was left between the plots. Randomization of this trial design was to limit variation (CV%) in results.
Trial site preparation
Prior to the trial, the area received high traffic as the camp provided a resting place for sheep requiring inoculations.The stand was cut to 50mm in height and left for disease to become re-established (approximately three weeks). The experimental area was isolated from any animal traffic for the duration of the trial period.
Treatments
Treatments included full, half and zero (control) application dosage rates for BIOSTART, ROOTSHIELD and PUNCH XTRA. Application rates of each treatment are tabulated below in Table 4.1. The zero applications, although not necessary in the trial for each
individual treatment, were included as further variables for comparison in terms of the efficiency of the treatments based on the individual replicate blocks. ROOTSHIELD was applied using 5Qwatering cans (one per treatment), with a pouring nozzle head of 6.2cm X 7.8cm, with spray holes 1mm apart. PUNCH XTRA and BIOSTART were applied using a pressurized spray bottle with a fine spray nozzle. These treatments were mixed with a water source obtained from the infield irrigation system.
Table 4.1. Application rates for the different treatments used in the 2000 trial to determine the efficiency of biological control agents for the control of Bipolarissp.
Treatment Treatment Application rate Manufacturer's
number administered annllcatlon rate
1 Water/ zero ROOTSHIELD Og/4e
2 Half recommended ROOTSHIELD 1.25g/4e 3009/5001 water/50m2
3 Full recommended ROOTSHIELD 2.5g/4e
4 Water/ zero PUNCH XTRA Ome/4e
5 Half recommended PUNCH XTRA O.04me/4e 750meha'
6 Full recommended PUNCH XTRA O.08me/4e
7 Water/ zero BIOSTART Ome/4e
8 Half recommended BlaSTART O.02me/4e 500meha-1
9 Full recommended BIOSTART O.05me/4e
Observations and analysis
The trial commenced on the 1st March 2000 with an initial rating before the first application of treatments. Five ratings and re-application of treatments followed at approximately 10 day intervals. Ratings were made according to Hall's (1991) rating scale of percentage leaf area infected byBipolaris sp. on P. clandestinum (Figure 4.1).
Ratings of a random sample of 10 leaf blades were made from each treatment plot. The sample area was also confined towards the plot centers, 40cm from the plot edges.
Ratings were limited as far as possible to new growth.
Dry matter (DM) % based on the wet and dry biomass of each plot was assessed.Wet biomass (g) samples were collected using a cutting disc measuring 30cm in diameter (area
=
706.9cm2of the 1m2plots). Samples again were limited to the center of the plots to eliminate the edge effect. Dry biomass (g) was determined by drying the wet samples for 48hrs at 60°C.The AUDPC was calculated, using Genstat 5 (Anon, 2000), for each treatment type dosage. Final percentage disease was also calculated as final percentage of disease (X1) over the initial disease (Xo) for each experimental plot. Statistical analysis of the AUDPC values, FD%, mean final wet and dry biomass and dry matter percentage were conducted using analysis of variance (ANOVA) in Genstat 5 (Anon, 2000). Due to the unbalanced trial design, ANOVA was determined using restricted maximum likelihood (REML analysis) to eliminate the treatment error between the various control treatments.
Figure 4.1 Disease severity rating scale for Helminthosporium leaf spot (Drechslera bicolour) , expressed as percentage leaf area infected on the first five leaves ofPennisetum clandestinum; where 1= 2%, 2=4%, 3=8%, 4=16% and 5=32% (Hall, 1991).
4.2.3.2 2002 trial Trial site
The trial site was moved due to the low disease levels following the 2000 trial. The area comprised a pure stand of P. clandestinum with heavy infestations of Bipolaris.
Trial design
The trial design was that of a randomised complete block design, with nine treatments and four replicates, laid out into 36, 1m2 plots. A border of 0.5mwas left between the plots. The trial design was a randomised complete block design. Replications were reduced from the 2000 trial, due to the new area being smaller. A 0.5m border was allowed. Randomization of this trial design was to limit variation (CV%) in results
Trial site preparation
Prior to the trial, the area received high traffic as it served as a walkway between camps.
The stand was cut to 50mm in height and left for disease to become re-established (approximately three weeks). The experimental area was isolated from any animal traffic for the duration of the trial.
Treatments
The treatments included full, half and zero (control) application rates for Bacillus 869, Trichoderma kd and PUNCH XTRA. Application rates of each treatment are tabulated below in Table4.2.Zero application rates were included for comparison, but were tallied as a single control for all treatments.
Trichoderma kd and Bacillus 869 were applied using 5~ watering cans (one per treatment), with a pouring nozzle head of 6.2cm X 7.8cm with spray holes 1mm apart.
PUNCH XTRA was applied using a pressurized spray bottle with a fine spray nozzle.
These treatments were mixed with a water source obtained from the infield irrigation system.
Table 4.2. Application rates for the different treatments used in the 2002 trial to determine the efficiency of biological control agents for the control of Bipolarissp . .
Treatment Treatment Application rate Manufacturer's
number administered application rate
1 Water/ zero Trichoderma kd Og/4e
2 Half recommended Trichoderma kd 2g/4e 1g/1e
3 Full recommended Trichoderma kd 4g/4e
4 Water/ zero PUNCH XTRA Ome4e
5 Half recommended PUNCH XTRA 0.04me/4e 750meha-1
6 Full recommended PUNCH XTRA 0.08me/4e
7 Water/ zero Bacillus 869 Og/4e
8 Half recommended Bacillus 869 2g/4e 1g/1e
9 Full recommended Bacillus 869 4g/4e
Observations and analysis
The trial commenced on the 19th March 2002 with an initial rating before the first application of treatments. Five ratings and re-application of treatments followed at approximately 10 day intervals. Ratings were made according to Hall's (1991) rating scale of percentage leaf area infected by Bipolaris sp. on P. clandestinum (Figure 4.1).
Ratings of a random sample of 10 leaf blades were made from each treatment plot. The sample area was also confined towards the plot centers, 40 cm from the plot edges.
Ratings were limited as far as possible to new growth.
Dry matter percentage based on the wet and dry biomass of each plot was assessed.
Wet biomass (g) samples were collected using a cutting disc measuring 30cm in diameter (area = n,-2= 706.9cm2of the 1m2plots). Samples again were limited to the center of the plots to eliminate the edge effect. Dry biomass (g) was determined by drying the wet samples for 48hrs at 60°C.
The AUDPC was calculated, using Genstat 5 (Anon, 2000), for each treatment type dosage. Statistical analysis of the AUDPC values, FD%, mean final wet and dry biomass and dry matter percentage was conducted using analysis of variance (ANOVA) in Genstat 5 (Anon, 2000).