Identification of the Causal Agent of Cocoa Pod Rot Disease From Various Locations

Cacao (Theboroma cacao L.) is an important estate commodity in Indonesia with high economic value. The interference of cocoa pod rot disease which was affected by Phytophthora palmivora Butl. resulted in the reduction of the quantity and quality of cocoa beans, with losses up to 44%. This research was aimed to figure out the variation in morphology of P. palmivora isolates from cacao. The research was carried out by collecting samples of cocoa pod with rot symptoms in several cacao growing areas in Java, then the pathogen was isolated and cultured on Potato Dextrose Agar (PDA) medium. The observation was performed on morphological characteristics of isolates macroscopically (colony shape) and microscopically (size of sporangium and chlamydospores). All tested isolates showed various colony shape such as stellate, cottony and irregular as well as sporangium varying from obpyriform, globose, ellipsoid, ovoid and distorted with various size between 30.8×21.9–65.5×46.5 µm in range.IntisariKakao (Theboroma cacao L.) merupakan komoditas perkebunan unggulan di Indonesia dengan nilai ekonomi tinggi. Gangguan penyakit busuk buah kakao yang disebabkan oleh Phytophthora palmivora Butl. mengakibatkan penurunan kuantitas dan kualitas biji kakao, dengan kerugian mencapai 44%. Penelitian ini bertujuan untuk mengetahui variasi morfologi isolat P. palmivora asal kakao. Penelitian dilakukan dengan mengambil sampel buah kakao bergejala busuk buah di beberapa area perkebunan kakao di Jawa, kemudian patogen diisolasi dan dikulturkan pada media Potato Dextrose Agar (PDA). Pengamatan dilakukan terhadap karakteristik morfologi isolat secara makroskopis (bentuk koloni) dan mikroskopis (ukuran sporangium dan klamidospora). Semua isolat yang diuji menunjukkan bentuk koloni seperti stellate, cottony, dan irregular serta sporangium yang bervariasi dari obpyriform, globose, ellipsoid, ovoid, dan distorted dengan ukuran bervariasi antara 30,8×21,9−65,5×46,5 µm.


INTRODUCTION
Cocoa pod rot which was caused by Phytophthora palmivora Butl. was the most important disease in cultivating cacao in Indonesia (Semangun, 2008). Yield losses resulted in the interference of cocoa pod rot disease in Indonesia reached 44% due to the reduction in quality and quantity of cocoa bean production (Rubiyo & Amaria, 2013). P. palmivora could attack almost whole parts of cacao plant, such as stem, flower cushion and leaves. The most destructive invasion occurred on pod since it directly related to yield loss (Opeke & Gorenz, 1974;Sri-Sukamto, 1985;Purwantara, 1990;Priyatmojo & Subandiyah, 1996;Rubiyo & Amaria, 2013). The initial symptom was spot on pod, then developed quickly and extended until covered whole surface of pod (Guest, 2007). On further infection, pathogen could invade the beans with symptom of blackening and wrinkling cocoa beans (Bowers et al., 2001). Wahyuno et al. (2007) reported that isolate of Phytophthora possessed various shape of sporangium, i.e. spherical to pear or lemon-shape with distinct papillae on the tip of sporangium. The investigation revealed that P. capsici from white pepper had the length of sporangium between 20 -88.8 µm, breadth of Submitted May 2, 2017;accepted September 20, 2017 17.5-55 µm, and length (l) to breadth (b) ration of sporangium between 0.9-2.8. This research was aimed to figure out the variation on morphological characteristics of P. palmivora isolates, causal agent of cocoa pod rot disease, from several locations of cacao plantations.

MATERIALS AND METHODS
This experiment was carried out in Laboratory of Plant Disease, Department of Crop Protection, Faculty of Agriculture, Universitas Gadjah Mada. Samples of P. palmivora-infected cocoa pod were collected from several cacao plantations, i.e. West Java (Sumedang and Cianjur), Central Java (Batang and Wonosobo), Daerah Istimewa Yogyakarta (Kulon Progo and Gunung Kidul), as well as East Java (Jember).

Preparaton of Isolates
Pathogen was isolated from part of cocoa pod showing symptom of pod rot. Symptomatic pods were washed with tapping water, and then surface-disinfected using ethanol 96%. The skin was peeled and pulp was cut in small size on adjacent part of healthy and diseased tissue, and then cultured on Potato Dextrose Agar (PDA) medium and incubated at temperature of 24°C. The emerged mycelium was then observed and directly identified under compound microscope. The growing mycelium was sub-cultured on the same medium to get the pure culture.

Molecular Identification
Molecular identification was aimed to ensure that the obtained isolate was P. palmivora. This analysis was conducted using PCR method with specific primers for P. palmivora (pal1s and pal2a) with target size of 650 bp (Chirapongsatonkul et al., 2015). DNA extraction of P. palmivora isolates was performed using CTAB method (Subandiyah, 2003).

Morphological Identification of Isolates
Morphological identification involved the observation of isolate characteristic macroscopically and microscopically. The observation of macroscopic morphology was performed on the growth of hyphae by daily measurement on the diameter of colony until 5 days after subculture and shape of colony on PDA medium. Meanwhile, the observation of microscopic morphology was performing by taking and putting the small cut of isolates from various locations on object glass which had been previously dropped with methylene blue solution and then warmed by passing them on Bunsen fire until melting and then covered with cover glass. Morphological characteristics were observed under compound microscope which was connected to Optilab software on computer. Shape and size of sporangium and chlamydospores were recorded.

Analysis of UPGMA
Morphological variation of tested isolates was analyzed with Unweighed Pair Group Method with Arithmatic mean (UPGMA) using NTSys program.

RESULTS AND DISCUSSION
Phytophthora palmivora was recognized having wide range of host plants such as coconut, cacao, papaya, durian, citrus, quinine and areca nut (Zentmyer, 1974;Agrios, 2005;Semangun, 2008). On cacao, the infection could occur in almost whole parts of plant, i.e. stem, flower, pod and leaf surface. Cocoa pod was the most susceptible part against the invasion of P. palmivora. Pathogen could infect at all stages of pod development, and immature pod was the most susceptible phage toward pathogen attack. The initial symptom of infection was spot on cocoa pod which would quickly develop within 14 days and then could extend covering whole pod's surface. Such symptoms were various on pod, i.e. from the tip of pod, base of pod close to stalk and irregular pattern. Further infection was indicated with the emergence of white powder which was the sporangium of P. palmivora on surface of diseased pod (Figure 1). At the same time, neighboring pod either on the same or different trees would express varying symptoms (Bowers et al., 2001;Guest, 2007;Rubiyo et al., 2008;Vanegtern et al., 2015).

Molecular Identification
The PCR test showed that DNA from all isolates could be amplified at 650 bp ( Figure 2). It proved that 13 collected isolates from rotting pods of 8 surveyed locations were P. Palmivora, the causal agent of black pod diseases on cocoa. The previous research of Chirapongsatonkul et al. (2015) also found that molecular identification of P. palmivora isolates with PCR method using specific primers of Pal1s and Pal2a could amplify the target DNA at 650 bp.

Macroscopic Characteristics
Macroscopic observation was conducted on the shape and diameter of colony which was cultured on PDA media at 5 day after subculture. There were variations in mycelia growth and shape of colony from each isolate which were revealed on Table 1. Table 1 showed that PTK.2811 isolate had the slowest growth of colony on PDA medium compared to other isolates, i.e. 37 mm at 5 day after subculture. This research grouped the variation in shape of colony into stellate, cottony and irregular (Figure 3).
Hyphae of P. palmivora on PDA medium was white and would grow downward or into medium, so that the colony looked thin on the surface of medium. In line with the research of Manti (2009), macroscopically colony of P. palmivora on PDA medium would grow slowly, round in shape with wavy margin, like cotton, white and elastic when it was cut using scalpel.

Microscopic Characteristics
The result of microscopic observation on morphological features showed that there was difference of species characteristics between sampled isolates from several locations, i.e. variation in size as shown in Table 2.
The variation in morphological characteristics of P. palmivora isolates either macroscopic or microscopic was not influenced by environmental factors and altitude of cacao plantations. Variation in shape of colony and size of sporangium was also characteristics of P. palmivora. These were stated as well by Erwin and Ribeiro (1996) that the difference in morphological characteristics of P. palmivora species depended on those isolates of species.
Based on UPGMA analysis using 7 morphological combinations, 13 isolates were grouped into two clusters with similarity of 70% ( Figure 5). The first group consisted of all isolates from Java Island excluded isolates from Cianjur (West Java) which was clustered into the second one together with one isolate from Gunung Kidul (Daerah Istimewa Yogyakarta). However, these clusters did not show the variablity on morphology of isolates against altitude and originating areas.

CONCLUSION
There were variations in type of rotting symptoms on pod, i.e. from the tip of pod, base of pod close to stalk and irregular pattern. On PDA media, colony growth of P. palmivora varied, i.e. stellate, cottony and irregular. P. palmivora had several shapes of sporangium varying from obpyriform, globose, ellipsoidal, ovoid and distorted with various size between 30.8×21.9-65.5×46.5 µm in range.