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Disease surveys:
During the first phase, three surveys were conducted to get information about the most important tomato diseases in Thailand. The surveys covered the area around Chiang Mai in the North of Thailand, the area along the borderline to Laos in the Northeast and the area around Cha Am in the South. As tomato production under protected cultivation is rare in Thailand and only possible with high fungicide input, mainly tomato production in open fields was checked in the surveys. Leaf miners, fruit borers and thrips were the most serious pests on tomatoes. A frequent occurrence of bacterial wilt was also reported. The most important leaf diseases were early blight (Alternaria solani) and late blight (Phytophthora infestans), the latter mainly in relatively cooler regions in the North. In the soil samples showing positive infestation, Pythium spp. were identified. In the warmer regions, P. aphanidermatum was the most relevant soil-borne pathogen.
Methodological experiments with the fungi:
In side experiments, methods for the inoculation, inoculum production, re-isolation and maintenance were tested for A. solani and P. aphanidermatum as well as later for Pseudocercospora fuligena.
Several inoculation tests were conducted with spore suspensions of A. solani isolates collected in the surveys and obtained from other countries. In theses tests, the effects of temperature and leaf age were tested using the tomato variety ‘King Kong 2’. In the greenhouse with natural conditions of 35°C, no characteristic symptoms of A. solani could be identified while at 25°C leaves showed symptoms which were more pronounced with increasing leaf age. From these experiments it was concluded that although early blight could be often found in surveys in Thailand, it would not be a problem under the warm conditions in the greenhouses at AIT.
Similarly, tests with P. aphanidermatum were conducted to find the inoculation method, the appropriate inoculation density, and methods for re-isolation. The best way to inoculate P. aphanidermatum was the method using agar pieces from Petri dishes, while the dipping method completely failed. As the root pathogen P. aphanidermatum shows no lesions on the upper part of the plant, methods for detection were compared and the baiting of the pathogen with potatoes turned out to be the easiest method.
In the second central experiment as well as in the second satellite experiment, the tomato plants in the greenhouses at the AIT showed symptoms of black leaf mold, caused by Pseudocercospora fuligena. Leaf samples with disease symptoms were collected and the fungus was isolated on PDA to be used in inoculation tests. Two different inoculation methods were compared, spraying a spore suspension or blowing the spores by air. By spraying the inoculum, the development of symptoms took approximately 4 days. This was 4 days faster than by blowing the inoculum onto the plants. With both methods the disease could be established. Thus the spread of the disease can be by wind-borne spores and not only by spores disseminated by splashing rain, running water and machinery, as stated in literature. Older leaves seemed to be more susceptible.
Satellite greenhouse experiments with the disease complex:
Altogether five satellite experiments were conducted with a disease complex in which Pythium disease was created by inoculating P. aphanidermatum in three densities. To establish early blight as leaf disease, A. solani was inoculated in the first two experiments. In the later experiments, black leaf mold (BLM) naturally occurred and it was necessary to apply fungicides to create different BLM levels.
In the first satellite experiment with tomato cultivar ‘King Kong 2’, symptoms of A. solani could not be identified. The visible damage was related to P. aphanidermatum, which could be successfully re-isolated from the soil. After three weeks, one third of the plants in the greenhouse was already dead. In addition, over 50% of the plants had lost more than 50% of the healthy tissue. This experiment was stopped after two months, because of a general fertiliser and plant nutrition problem in all greenhouses. As the inoculation of A. solani did not result in an early blight epidemic, this inoculation had no significant effect on fresh biomass and root weight of tomato plants. In contrast, the increasing inoculation densities of P. aphanidermatum reduced both weights, although not significantly because of the high plant variability. With respect to yield, the reducing effect of P. aphanidermatum was also observable (except for the number of fruits in the treatment without A. solani). Surprisingly the number of fruits and the total weight were higher on plants inoculated with A. solani compared to the non-inoculated plants, but only in the treatment without P. aphanidermatum inoculation.
In the second experiment, the variety ‘New King Kong’ was used, a semi-determinate variety, due to an error of the seed company. The inoculations with A. solani and P. aphanidermatum were successful which was shown by re-isolation of both fungi. The leaves inoculated with A. solani showed necrosis or wilting, but no lesions could be observed on non-inoculated leaves. Obviously, no secondary infections occurred and again no early blight epidemic could be observed. Nevertheless, the inoculation with A. solani resulted in a reduction of fresh biomass, leaf area and fruit weight, while the effect on root weight was variable. The effects of the inoculum concentrations of P. aphanidermatum were not uniform, although in general the negative effects of higher spore concentration could be seen, especially on fruit weight and number of fruits in the treatment with A. solani inoculation. In this experiment, symptoms of black leaf mold (BLM) caused by Pseudocercospora fuligena (= Cercospora fuligena) occurred and the progress of BLM, expressed as disease incidence of plants, followed an S-shaped curve, reaching 100 % at 63 days after transplanting.
The third experiment was carried out with variety ‘King Kong 2’ using a new inoculation method for P. aphanidermatum. However, the plants of this satellite experiment died rapidly. One week after starting the experiment, over 60% of the plants were almost dead. Symptoms of wilting and damping- off were related to P. aphanidermatum that could successfully be re-isolated from the soil. Because of this damage, the experiment was stopped and another one was started immediately.
In the fourth experiment, conducted again with variety ‘King Kong 2’, BLM naturally occurred in the greenhouse. Therefore, no inoculation was needed, but one half was sprayed with fungicides to slow down the BLM epidemic there. In both parts, P. aphanidermatum was inoculated in 3 different densities in the soil. With a run-test it was shown that the BLM diseased plants were randomly distributed within the rows, except of one row in the third observation. The BLM incidence in the outer rows was slightly higher so that a weak disease gradient could be observed from the sidewall to the centre across rows. The BLM progress curves (with disease incidence) in the three Pythium inoculations with and without BLM control were similar, both groups reaching 100% around 70 days after transplanting (dat). The three curves without fungicide sprays, differing in P. aphanidermatum inoculation, were steadily increasing, while the curves in the sprayed variants did not increase or even decreased between 28 and 49 dat due to the fungicide application. However, the BLM severity in both groups (with and without control) remained at a low level (below 1 %) until 70 dat. Thereafter, the disease severity increased up 8 % on plants without BLM control, whereby the severity was higher on plants inoculated with high density of P. aphanidermatum compared to plants not affected by root rot. The treatments with low and high inoculum levels of P. aphanidermatum reacted very similar with respect to root weights and shoot dry weights, which were significantly lower than in the control (without Pythium). On the other hand, the control of BLM led to a significant increase in root weight (at 28 and 84 dat) as well as in shoot dry weight (at 56 and 84 dat). It could be concluded that the inoculation with P. aphanidermatum influenced the plant and yield data stronger than the fungicides sprays against BLM.
The experimental set-up of the fifth satellite experiment, which is still ongoing, was identical to the fourth one. During the first three weeks after transplanting, the experiment was dominated by the effect of the inoculation with P. aphanidermatum. Several plants died rapidly after inoculation. On average, a loss of 30 % of the plants was observed. The first symptoms of BLM appeared 21 dat. All plants showing symptoms were recorded in order to generate maps of diseased plants to follow the spread of BLM within the greenhouse. However, the statistical analyses of these spatial patterns were difficult, because several tomato plants were heavily damaged as a result of the P. aphanidermatum inoculation and therefore subsequently removed. Therefore, the distances between plants within rows were no longer uniform preventing the usual spatial analyses for rows like run and doublet analysis.
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