Abstract
3.2 Trial 1 - The effect of dosage rates of Eco-T®, in a recirculating (closed) hydroponics system, on Pythium control and plant growth
3.2.2 Materials and methods
Speedling®-24's were planted with lettuce seed (Mayford - All Year Round) in pine bark medium. The seeds were left to germinate for two days in the potting shed. Thereafter, one tray was placed into each of the 36 horizontal mini troughs (Fig. 3.2.2.A).Three replicates of each of the 11 treatments (Table 3.2.2.A) were carried out and these were arranged in a randomised block design.
Table 3.2.2.A Treatments used in Trial 1
Treatment Pythium added Trichoderma (g/l) Trichoderma (conidiaIQ)
1 no 0 0
2 yes 0 0
3 yes 1 2x109
4 yes 0.5 1x109
5 yes 0.25 5x108
6 yes 0.125 2.5x108
7 no 1 2x109
8 no 0.5 1x109
9 no 0.25 5x108
10 no 0.125 2.5x108
11 no 0.0625 1.25x108
The Eco-~ formulation used consisted of 2x109 conidia/g. The reservoirs for each trough contained 7Q of nutrient solution and Trichoderma was added directly to the reservoirs along with fertilizer. Plants were inoculated with Pythium by means of 25mm2 agar blocks taken from a one week old culture grown on potato dextrose agar (PDA) The agar blocks containing Pythium growth were placed on the surface of the growing medium next to each emerging seedling 24 hours after Eco-~ treatments.
The Pythium isolate used in this and all subsequent trials was isolated from a commercial hydroponic system where both lettuce and strawberry plants were being grown. Samples of growing medium (50mQ) were placed in 250mQ beakers to which 150mQ of distilled water was added. Citrus leaf discs were floated on the surface as a selective bait. Leaf discs were removed after two days, dried on paper towel, and placed onto water agar. Fungal growth was identified microscopically after five days as being a Pythium species. This identification was later confirmed by the Plant Protection
Research Institute4 (PPRI) as being Pythium myriotylum Drechsler (PPRI accession number 04169). The isolate was stored both on agar plugs in sterile distilled water and in double autoclaved sand to prevent attenuation.
Water temperature was maintained at ± 26°C. Ocean Agriculture 3: 1:3 (38) Complete fertilizer was used (1 g/l), with the nutrient solution maintained at an electrical conductivity (EC) of 1.8 ms (millisiemens). pH was amended once a week using nitric acid or potassium hydroxide to maintain the pH at 6.0. Plants were harvested once a visual difference in plant size was observed between treatments (24 days).
Once the trial was completed, all 36 troughs were emptied and the troughs and components washed down with, or rinsed in, a solution of Sporekill® (a quaternary ammonium compound (QAC) sterilant)5. The troughs were then reassembled and the experiment repeated. However, a few important changes were made. When planting, two seeds were placed in each cell of the speedling tray. The seeds were then allowed to germinate in the potting shed for two days, as before. They were then allowed to grow for 10 days in the seedling greenhouse. On transferring the trays to the hydroponic troughs, the trays were thinned out, or made up, to 24 plants each. This provided a more equal and comparable base between replicates at the start of the trial. In the repeat trial the formulation of Eco-T®, supplied by Plant Health Products (Pty) Ltd, had been diluted to 5x108 conidia/g on the basis of earlier dose trials. An extra dose(0.0625g/Q
=
3.125x1 07conidia/Q) was used in the biocontrol treatments, giving 12 treatments in total.In both trials, total shoot wet weight was recorded for each replicate and statistical analysis was done using the SAS system for Windows 98, Version 6.1. Analysis of variance (AN OVA) and the Student-Newman-Keulstestwere conducted. The mean wet weight for each treatment was calculated and graphed.
4ARC-Plant Protection Research Institute, Private Bag X134, Pretoria, 0001, South Africa 5Hygrotech Seeds, P.O. Box 21880, Mayors Walk, 3208, South Africa.
3.2.3. Results
In Trial 1a. (Fig. 3.2.3.A and Table 3.2.3.A), significant yield losses were achieved by inoculation with Pythium (inoculated control). Significant levels of biocontrol were recorded at all doses when compared with this inoculated control. However, at all doses except0.25g1Q,yield was still below that of the uninoculated control. At 0.25glQyield was significantly higher than in the uninoculated control, although the difference was only approximately three grams in mean total wet weight.
In Trial 1b. (Fig. 3.2.3.8 and Table 3.2.3.8) the same trends were recorded with the only difference being that at 0.5g1Q Trichoderma resulted in yields not significantly different to the uninoculated control.
In growth stimulation Trial 1a. (Fig. 3.2.3.C and Table 3.2.3.C) no significant changes in yield were recorded under the different treatments.
In Trial 1b the same trends can be seen when comparing Figs. 3.2.3.C and O. In this repeat trial, however, statistically significant differences were obtained. At Trichoderma doses of 1 and 0.5 glQyield was seen to be significantly lower than for the uninoculated control (Table 3.2.3.0). This was a significant yield reduction response. No significant growth stimulation was recorded at any of the doses.
60
~50.9
-
..s=
'(i)Ol40 3:
Q5 3: 30 cro
Q)
~ 20
A
B ~
D '-' D
E
- -
10 control (u) 1g/l 0.25g/l
control(i) 0.5g/l 0.125g/l
Treatments
Fig. 3.2.3.A - Biocontrol dose response Trial 1a - Trichoderma vs. Pythium on lettuce. Treatments with the
same letter are not significantly different at p= 0.05
Key to Figs. 3.2.3.A and B
Control (u) = Uninoculated control
Control (i)= Inoculated control (inoculated with Pythium) All other treatments were inoculated withPythium and treated with the dose of Eco-T® shown ing/Q
0.0625 0.125g/l
A
0.25g/l 0.5g/l
Treatments
1911 control (i)
n 0
I~
'"'
n ~ L
b
90 :§80
'w~70
~Q) 60
~ 50
~co 40 30control (u)
Fig. 3.2.3.B - Biocontrol dose response Trial 1b - Trichoderma vs. Pythium on lettuce. Treatments with the
same letter are not significantly different at P= 0.05
55 - , - - - ,
25 control (u)
19n
0.5g/l
0.25g/l
Treatments
0.125g/l
0.0625gn
Fig. 3.2.3.C Growth stimulation dose response - Trial la.
Treatments with the same letter are not significantly different at P= 0.05
Keyto Figs. 3.2.3.C and D Control (u)
=
Uninoculated controlAll other treatments received only the dose of Eco-T®
shown ing/Q
100 :§ 80
-
.!:
Ol 60
·iD 3:
Q) 3: 40
c
Cl) 11l
:2 20
0 control (u) 19n
0.5g/l 0.125gn
0.25gn 0.0625gn
Treatments
Fig. 3.2.3.D Growth stimulation dose response - Trial lb.
Treatments with the same letter are not significantly different at P = 0.05
Table 3.2.3.A Summary of results for Trial 1a - biocontrol dose response
treatment Pythium Eco-~(gte) mean wet rank Student- weight (g) Newman-Keuls
(SNK) grouping
1 no 0 50.67 2 b
2 yes 0 34.00 6 e
-
3 yes 1 44.33 5 d
4 yes 0.5 47.67 3 c
5 yes 0.25 53.33 1 a
6 yes 0.125 44.67 4 d
f=114.36 P=0.05 CV (%) = 2.38
* Treatments sharing the same Student-Newman-Keuls (SNK) letter do not differ significantly
Table 3.2.3.8 Summary of results for Trial1b - biocontrol dose response
treatment Pythium Eco-T® (gIQ) mean wet rank SNK
. weight (g) grouping
1 no 0 86.05 3 b
2 yes 0 63.58 7 e
3 yes 1 76.32 6 d
4 yes 0.5 87.25 2 b
5 yes 0.25 88.77 1 a
6 yes 0.125 81.2 4 c
7 yes 0.0625 80 5 c
f = 319.94 P=0.05 CV (%) = 1.04
* Treatments sharing the same Student-Newman-Keuls (SNK) letter do not differ significantly
Table 3.2.3.C Summary of results for Trial 1a - growth stimulation dose response treatment Eco-~(gIQ) mean wet rank SNK grouping
weight (g)
1 0 50.67 1 a
2 1 40.33. 6 a
3 0.5 41.00 5 a
4 0.25 46.33 4 a
5 0.125 47.33 3 a
6 0.0625 47.67 2 a
F = 1.86 P=0.05 CV (%)
=
11.33* Treatments sharing the same Student-Newman-Keuls (SNK) letter do not differ significantly
Table 3.2.3.D Summary of results for Trial 1b - growth stimulation dose response
Treatment Eco-T (gIQ) Mean wet rank SNK grouping weight (g)
1 0 86.33 2 a
2 1 45.67 6
c
3 0.5 71.33 5 b
4 0.25 75.00 4 ab
5 0.125 76.33 3 ab
6 0.0625 86.67 1 a
F = 23.08 P=0.05 CV (%) = 7.36
* Treatments sharing the same Student-Newman-Keuls (SNK) letter do not differ significantly
3.2.4 Discussion
In both trials Eco-T® was seen to achieve significant disease control at all doses, when compared with the inoculated controls (Tables 3.2.3.A andB). However, it is evident that at high concentrations, Trichoderma does not function at an optimum. In both trials
o
.25g/Qgave the best results, although in the second trial, in which the formulation was diluted four fold, O.5g/Q functioned noticeably better than in the first. This is advantageous for manufacturers, as it allows dilution of the product while achieving better control, increasing the likelihood for economic competitiveness and success.No significant growth stimulation was recorded (Figs. 3.2.3.C and 0). Instead, Trichoderma applied in the absence of the pathogen resulted in a decrease in yield at most doses. These reductions in yield were significant in the repeat trial at 1 and 0.5g/Q (Table 3.2.3.0). A possible explanation for this phytotoxicity is provided by Cutleret al (1986) and Cutler and Jacyno (1991) who studied metabolites, with phytotoxic activities, produced by strains of Trichoderma. These included 6-pentyl-cx-pyrone (Cutler et al., 1986) as well as (-)harzianopyridone, Koninginin A and Koninginin B (Almassi et al., 1991, cited by MacKenzie et al., 2000).
Another possible explanation could be related to the use of fertilizer containing high levels of ammonium nitrogen (NH4-N). In a medium like composted pine bark, much of this NH4-N would normally be converted to nitrate nitrogen (N03:"'N) by nitrification bacteria. Trichoderma shows a preference for NH4-N (Wakelin et al.) 1999) and could, in large artificial populations, compete with these bacteria and reduce the nitrification process. This would result in an abnormally high level of NH4-N in the root zone and could lead to ammonia toxicity. This theory is covered in greater depth in Chapter 5.
A further mechanism could be linked to the secretion of plant growth hormones by Trichoderma. This would have obvious benefits if Trichoderma were to be considered as a mycorrhizal fungus (i.e., increased growth of the host allows for increased growth . of the mycorrhizal organism). In large artificial populations it might be possible thatthe production of growth hormones byTrichoderma would be in such large concentrations that it would result in inhibitory responses.