Effects of humidity, temperature and inoculum density under controlled environment and fungicide application under field conditions on the development of Cercospora leaf spot of Swiss chard

Abstract

Cercospora leaf spot (CLS), caused by Cercospora beticola is an important foliar disease of Swiss chard in Botswana. Regular outbreaks of the disease in Botswana is a major cause of concern and thus have necessitated the use of fungicides under field conditions in order to control the disease and bring the crop to maturity. The objectives of the study was (1) to analyse the genetic diversity of C. beticola isolates from Swiss chard collected from southern Botswana, (2) to determine the effect of humidity temperature and inoculum concentration on plant disease development, under controlled environment and (3) to assess the effectiveness of locally available fungicide in managing CLS of Swiss chard under field conditions in Botswana. Genomic DNA extraction from C. beticola mycelia was done using the MasterPure Yeast DNA purification Kit and PCR amplification utilized ITS1 (5’-TCCGGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) primers in a Techne thermocycler and the phylogenetic tree of ITS rDNA was constructed using version 6.06. Phylogenetic analyses of 87 isolates revealed that C. beticola isolates clustered in four major classes, which could not be differentiated by the sampling location. Similarly genetic analysis revealed high genetic diversity of C. beticola strains, with diversity accounted for by within population diversity of Swiss chard isolate (greatest pairwise distance = 0.005). The results presented herein underscore the importance of genetic diversity of pathogens which may be important in targeted control and management of plant diseases. Effect of humidity, temperature and inoculum concentration on disease development over a 4-week period was conducted in the Department of Agricultural Research (DAR) Laboratory, using a modified egg incubator as a controlled environment growth chamber. The humidity and temperature treatments were run separately and sequentially each for 28 days where the incubator was set at 50%, 70% and 90% relative humidity (RH) and at 25°C and at 15° C, 20°C, 25°C and 30°C, respectively, and plants inoculated with 0x104, 0.1x104, 1.2x104, 2.3x105 and 2.3x106 spores/mL using a hand sprayer. A completely randomised design for the three-factorial experiments (RH x Inoculum Concentration x Time and Temperature x Inoculum Concentration x Time), with ten replicate plants was used. Number of spots per leaf for inoculated plants at 25°C increased significantly (P≤ 0.05) when plants were incubated at 2.9) RH, respectively. As inoculum increased from 0 to 2.3x106 spores/mL the number of leaf spots also significantly increased from 0.4 to 2.9 spots per leaf. The highest number of spots per leaf was recorded on plants inoculated with 2.3x106 spores/mL and incubated at 90%RH (4.4) followed by 2.3x105 spores/mL at 90%RH and 2.3x106 spores/mL at 70%RH. For each spore concentration the amount of disease significantly increased with increase in humidity. At 90%RH disease development was highest at 25°C (2.9 spots/leaf)) followed by 30°C (2.5 spots/leaf), 20°C (1.9 spots/leaf) and 15°C (no spots). Averaged across inoculum concentrations and humidity number leaf spots significantly increased from 0.760, 1.680, 2.225, to 2.580 spots/leaf from week 1 to 4, respectively. Similarly the interactions of time with inoculum concentrations and humidity resulted in significant increases in number of leaf spots per plant from week 1 to week 4. The results showed that low temperatures and low humidity which prevail during the winter in Botswana are not conducive while temperatures and humidity during the rainy season are more conducive to CLS disease development. The study confirmed that as RH increases, CLS numbers also increase. Relative humidity assessed alone or interacting with either inoculum concentration or sampling time increased as humidity increased. Cercospora leaf spot increased significantly (P ≤ 0.05) from 0.795, 1.855 to 2.86 at 50%, 70% and 90% RH respectively. The interaction of humidity with % was 0.400 CLS for the 0x 104 concentration compared with 1.250 for the 2.3x106 concentration. The 70% RH recorded 1.325 CLS for the 0x104 concentration and 1.855 CLS for the 2.3x106 concentration. The 90%RH had higher value of CLS 1.725 for the 0x104 concentration compared with a 4.350 at 2.3x106 concentrations. These values showed that as RH and inoculum concentration increased CLS also increased significantly at P ≤ 0.05. Moreover Sampling time alone or interacted with RH showed an increase in CLS as both humidity and sampling time increased significantly P ≤ 0.05. In addition temperature assessed alone or interacted with inoculum concentration or sampling time significantly increased CLS development as temperature increased from 20°C to 25°C from 1.925 to 2.835 respectively. However at 15°C there was no CLS development (0.000) and at 30°C CLS development reduced significantly to 2.485 at P ≤ 0.05. As Inoculumconcentration increased CLS also significantly increased from 1.031, 1.325, and 1.594 to 2.769 for the 0x104, 0.1x104, 1.2 x104, 2.3x105, and 2.3x106 inoculum concentrations per ml. Cercospora leaf spot increased significantly when assessing the effects of Sampling time (7, 14, 21, and 28 days) from 0.760, 1.680, 2.225, to 2.580 respectively showing that the longer the plants are kept the more disease they develop. Furthermore when sampling time is interacted with temperature, the highest means are realised at 28 days for the 25°C, 30°C, 20°C and lastly 15°C where no CLS developed. Assessment of locally available fungicides for the control of CLS was carried out in Sebele using 5m x 2m plots arranged in a randomized complete block design (RCBD) with three blocks and seven fungicide treatments and untreated control Swiss chard. Seedlings were transplanted into the plots spaced 40cm inter-rows and 30cm between plants and natural infection was used. Once symptoms were observed plants were sprayed at 2-week intervals for 12 weeks with distilled water, copper oxychloride; benomyl + mancozeb; mancozeb + copper oxychloride; benomyl; benomyl + copper oxychloride; mancozeb; benomyl + mancozeb + copper oxychloride using recommended rates. Where more than one fungicidewas used, the fungicides were applied at alternating intervals. The average number of leaf spot per leaf was recorded two weeks after each fungicide application and samples were collected prior to spraying. Data was subjected to one-way analysis of variance and means separated using LSD test at p=0.05. Number of spot/leaf and percentage disease reduction in all treatments involving benomyl were 29.3±5.5 and 85%, respectively which was significantly lower than on those treatments involving mancozeb and copper oxychloride used alone or in combination which were 50.85±9.5 and 74.2%, respectively while the had 196.9 spots/leaf. This study confirms that when benomyl is alternated with copper oxychloride, it is much more effective in managing Cercospora leaf spot than mancozeb or copper oxychloride used alone or alternated. Key words: Cercospora beticola, Swiss chard, Cercospora leaf spot (CLS), ITS PCR, genetic diversity, Humidity, Inoculum, Environment, Fungicides, control, Botswana.