Journal of Agricultural Engineering and Biotechnology
May 2015, Vol. 3 Iss. 2, PP. 58-62
Morphological Variation of Peronosclerospora maydis, the Causal Agent of Maize Downy Mildew from Different Locations in Java-Indonesia Fitri Widiantini1, Endah Yulia2, Tiara Purnama3 1, 2, 3
Department of Plant Pests and Diseases, Faculty of Agriculture, Universitas Padjadjaran Jl. Raya Bandung-Sumedang KM 21, Indonesia 45363 1
[email protected]
Abstract-Maize downy mildew is one of the major constraints for maize production in Indonesia. Peronosclerospora maydis is known as the causal agent of downy mildew in Java, Indonesia. The use of fungicides yielded different reactions to the disease at various locations in Indonesia. The aim of this study was to look for evidence of variation in P. maydis by comparing morphological characteristics of downy mildew pathogen in several locations at Java, Indonesia. Samples were collected from eight locations in Java. The result showed that P. maydis was the causal agent of downy mildew in all locations. The shape and size of the conidia and conidiophores varied between locations, with the sizes being smaller compared to previous descriptions. These morphological variations indicate the presence of genetic variation. However, this needs further confirmation. Keywords- Maize Downy Mildew; Peronosclerospora Maydis; Morphology Variation
I.
INTRODUCTION
Maize (Zea mays L.) is the most productive and widely cultivated crop in the world. Maize cultivation covers more than 100 million hectares in 125 countries [1]. By 2050, maize demand is predicted to double [2]. However, plant diseases offer a major constraint for maize production. One of the causal agents for maize yield loss is the presence of maize downy mildew which caused by oomyctes of Peronosclerospora. The disease occurs at every stage of maize plant growth, from seedling until harvesting. Downy mildew has the potential to reduce up to 30% of the total maize production worldwide [3]. In Indonesia, downy mildew causes a yield loss of 50-100% on susceptible varieties [4]. Maize has been reported to host eight species of Peronosclerospora ie: P. heteropogoni, P. maydis, P. miscanthi, P. philippinensis, P. sacchari, P. sorghi, P. spontanea and P. eriochloae [5]. The genus of Peronosclerospora has two additional new members which were found in Australia. P. australiensis, the pathogen of maize downy mildew and P. sargae, the pathogen of sorghum downy mildew. Maize downy mildew in Australia had previously been known to be caused by P. maydis. Until recently, maize downy mildew in Australia has been found to be caused by P. australiaensis. This species differentiation was due to the difference of host range and oospore production between P. maydis and P. australiaensis [6]. P. maydis is known as the causal agent of maize downy mildew in Indonesia. However, recent findings showed that maize downy mildew in Indonesia is caused by three species of Peronosclerospora, which are P. maydis, P. sorghi and P. philippinensis [7, 8]. These three species are spread throughout several maize cultivation regions in Indonesia. P. maydis infection was reported at a maize plantation in West Java, Central Java and Lampung. P. philippinensis is found in South Sulawesi, North Sulawesi and Gorontalo, whilst P. sorghi mostly infects maize plantations in North Sumatera [7]. Despite the introduction of resistant varieties and the use of metalaxyl to control downy mildew, the disease is still a major problem of maize cultivation. The use of resistant varieties to control plant disease is considered as the most effective method. However, resistant varieties are not always available or when it is available, its resistance is often not long-lasting [9]. Furthermore, a recent outbreak of sorghum downy mildew in Texas indicated that the pathogen has become resistant to metalaxyl [10]. A similar situation was also noted in Indonesia, where metalaxyl was found unable to control maize downy mildew in East Java. Disease severity of 78% was detected in a plot trial treated with metalaxyl at the concentration of 7.5 g/kg seed compared to disease severity of 83% on untreated plot [11]. These findings indicated that resistance of Peronoscleropora spp. to metalaxyl continues to gradually progress. Different reactions of the maize downy mildew to fungicide use was detected in different locations. Metalaxyl was found ineffective to control maize downy mildew in West Borneo [12] and Kediri, East Java [11]. One possible explanation for this degree of variation in effectiveness to metalaxyl has been suggested due to the presence of P. maydis variability. An original description of P. maydis (Racib) by C. G. Shaw (1978) reported that conidia were spherical to subspherical in shape with dimensions of 17-23 µm x 27-39 µm, whilst conidiophores were formed in clusters of 150 to 550 µm in length [13]. The aim of this study was to look for evidence of P. maydis variation by comparing morphological characteristics of the downy mildew pathogen at several locations in Java Island, Indonesia.
- 58 DOI: 10.18005/JAEB0302002
Journal of Agricultural Engineering and Biotechnology
II.
May 2015, Vol. 3 Iss. 2, PP. 58-62
MATERIALS AND METHODS
A. Maize Leaf Sample Collection The method for collecting conidia of Peronoscleropsora was performed by following method [7] as described below. Maize leaves infected at the vegetative stage by downy mildew were selected. Preferably maize plants 30-50 days old were selected. The infected third leaf from the top was selected for sample preparation and further analysis. It was cut at the basal end and kept in zip lock plastic bag. B. Slide Preparation The third infected leaf from infected maize plants collected from the field were thoroughly washed under running water. The leaves were then air-dried and placed in glass jars containing a 2% glucose solution with at least 1-2 cm of the leaf base soaked in the sugar solution. The leaves were covered by plastic to keep moist and kept at room temperature until around 9 pm (maximum storage 6 hours). The leaves were then carefully washed to remove the sugar solution and placed inside zip lock plastic bags with the surface of the leaf facing up. These were placed outside in the yard in the same orientation until 4 am. The leaves was brought back inside, removed from the zip lock bags and placed on a table with the leaf surface was facing up. Using a flash light, the surface of the leaf was observed for the presence of conidia and conidiophore of Peronosclerospora as indicated by the presence of white powder on the leaf surface. The conidia and conidiophore were collected by attaching clear sticky tape to the leaf surface and placed onto a glass object containing a drop of methylene blue. The clear sticky tape was sealed using clear nail polish and kept for further microscope analysis. The oomycete of downy mildew was characterized by measuring the conidia and conidiophore dimensions using an ocular micrometer and stage micrometer. The first 25 undamaged conidia and conidiophore found under the microscope were assessed. The dimensions of conidia (length and width, x400) and conidiophore (basal cell-branching and branch length, x200) were measured. III. RESULTS AND DISCUSSION Samples were collected from eight locations of maize plantations in Java. One sample was collected from West Java (WJ), three samples collected from Central Java (CJ1, CJ2 and CJ3) and four samples were collected from East Java (EJ1, Ej2, EJ3 and EJ4) (Table 1). The maize downy mildew was causing significant problems at those locations, especially on sweet corn varieties. The same cultivar of sweet corn was planted in all locations. Seed treatment with fungicide to control downy mildew was implemented at most of the locations with the exception of samples collected at West Java. However, the use of fungicide did not seem to affect the disease infection. TABLE 1 LOCATIONS OF MAIZE SURVEYED TO STUDY MORPHOLOGICAL VARIATIONS IN DOWNY MILDEW FUNGUS
No.
Location
Host
Code
1.
Jatinangor-West Java
Sweet corn
WJ
2.
Jogonalan Klaten-Central Java
Sweet corn
CJ1
3.
Boyolali-Central Java
Sweet corn
CJ2
4.
Purwodadi-Central Java
Sweet corn
CJ3
5.
Palemahan Kediri-East Java
Sweet corn
EJ1
6.
Pagu Kediri-East Java
Sweet corn
EJ2
7.
Pare Kediri-East Java
Sweet corn
EJ3
8.
Papar Kediri-East Java
Sweet corn
EJ4
Morphological characteristics of the maize downy mildew pathogen are presented at Table 2 and shows that P. maydis was the causal agent of maize downy mildew at all locations. Conidia and conidiophores from all of samples displayed the typical morphology of P. maydis. Conidia of P. maydis were spherical to subspherical in shape with dimensions of 17-23 µm x 27-39 µm, whereas the conidiophores formed clusters 150 to 550 µm in length with dichotomous branching two to four times. The presence of oospores is not reported [13]. The results of the study showed that the size of conidia and conidiophores collected varied between locations. Conidia dimensions of P. maydis isolates WJ, CJ1, CJ2 and CJ3 were relatively smaller than isolates EJ1, EJ2, EJ3 and EJ4. Although, the conidia and conidiophores structure resembled P. maydis characteristics, the dimension of conidia and conidiophores of all isolates were smaller compared to the description given by CIMMYT [13]. Morphological characteristics of Peronosclerospora can vary depending on the host species or cultivar and environmental conditions [14]. The maize cultivar in all the locations where the samples were collected was similar. Therefore, the morphology variation of P. myadis may be influenced by the environmental conditions.
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Journal of Agricultural Engineering and Biotechnology
May 2015, Vol. 3 Iss. 2, PP. 58-62
TABLE 2 MORPHOLOGICAL CHARACTERISTICS OF P. MAYDIS FROM VARIOUS MAIZE CULTIVATION REGIONS IN INDONESIA
No. 1.
Location WJ
Morphology Conidia shape and dimension: subspherical (1017.5 x 17.5-22.5 µm) Conidiophore: 150-220 µm, tapering toward basal, dichotomously branched (2-4 branches), hyaline. Cell wall: one thin layer P. maydis
2.
CJ1
Conidia shape and dimensions: subspherical (1013.75 x 17.5-27.5 µm) Conidiophore: 220-450 µm, tapering toward basal, Dichotomously branched (2-4 branches), hyaline. Cell wall: one layer, thin P. maydis
3.
CJ2
Conidia shape and dimensions: spherical/ subspherical (10-15 x 17.5-22.5 μm). Conidiophore: 200-350 µm, tapering toward basal, dichotomously branched (2-4 branches), hyaline. Cell wall: thin/one layer P. maydis
4.
CJ3
Conidia shape and dimension: spherical/ subspherical (11.25-15 x 12.5-20 μm). Conidiophore: 200-340 μm, long and tapering toward basal, dichotomously branched (2-4 branches), hyaline. Cell wall: thin/one layer P. maydis
- 60 DOI: 10.18005/JAEB0302002
Picture
Journal of Agricultural Engineering and Biotechnology
May 2015, Vol. 3 Iss. 2, PP. 58-62
No. 5.
Location EJ1
Morphology Conidia shape and dimension: spherical (12.5-17.5 x 12.5-17.5 μm). Conidiophore: 110-180 μm, tapering toward basal, dichotomously branched (2-4 branches), hyaline. Cell wall: thin/one layer P. maydis
6.
EJ2
Conidia shape and dimension: subspherical/ spherical (12.5-15 x 12.5-20 μm). Conidiophore: 170-250 μm, tapering toward basal, dichotomously branched (2-4 branches), hyaline. Cell wall: thin/one layer P. maydis
7.
EJ3
Conida shape and dimension: spherical (12.5-17.5 x 15-22.5 μm). Conidiophore: 130-200 μm, tapering toward basal, dichotomously branched (2-4 branches), hyaline. Cell wall: thin/one layer. P. maydis
8.
EJ4
Conidia shape and dimension: spherical (12.5-15 x 12.5-20 μm). Conidiophore: 150-190 μm, tapering toward basal, dichotomously branched (2-4 branches), hyaline. Cell wall: thin/one layer. P. maydis
Picture
Conidia and conidiophores variability among isolates of P. maydis from different regions were also suggested due to the presence of genetic variation between P. maydis isolates. Bock et al. [15] reported morphological variability between P sorghi isolates from some locations in Africa. The variability was also shown at the level of pathogenicity; as some isolates were - 61 DOI: 10.18005/JAEB0302002
Journal of Agricultural Engineering and Biotechnology
May 2015, Vol. 3 Iss. 2, PP. 58-62
more pathogenic than others. Furthermore, Perumal et al. [10] showed that P. sorghi collected from 14 regions had genetic variability following analysis by AFLP (Amplified Fragment Length Polymorphism). The use of metalaxyl as a seed treatment was able to reduce the infection of sorghum downy mildew (SDW) by P. sorghi. However, a recent outbreak of SDW in Texas demonstrated that seed treatment with metalaxyl was no longer effective in inhibiting the infection of P. sorghi. AFLP was able to show the emergence of a new pathotype of P. sorghi resistant to metalaxyl [10]. In this research, genetic variability between P. maydis needs to be determined since there was an indication that this was occurring. The use of metalaxyl has been reported to be unable to inhibit the downy mildew infection. In Kediri-East Java, fungicides with metalaxyl as the active ingredient used at a high concentration of 7.5 g/kg was not able to inhibit disease infection. The downy mildew disease at the plot trial treated with fungicide was not significantly different to the control plot. IV. CONCLUSION Maize downy mildew infecting several maize plantations in Java Island-Indonesia was caused by P. maydis. Despite the similarity of the maize cultivars infected by the disease, conidia and conidiophores sizes varied between locations and the sizes were relatively smaller compared to previous descriptions. This indicates that environmental conditions may have played a major role in the presence of genetic variation of P. maydis. REFERENCES
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