Effect of Calcium Polysulfide Applications on Severity of Cocoa Pod Rot (Phytophthora palmivora Butl.) and Fungal Diversity

Indonesian government gives a high priority for the development and revitalization of cocoa production. In 2012/2013, production of cocoa in Indonesia reached 410,000 ton from 1,774,500 ha (about 231 kg ha-1), lower than Ghana which is 835,000 ton from 1,600,300 ha (about 521 kg ha-1). One of the constraints of cocoa production in Indonesia is pod rot disease caused by Phytophthora palmivora, which can reduce about 90% of production during wet season. Calcium polysulfide was suggested as one of the potential, cheap and save substance to control P. palmivora. The purpose of this study was to evaluate the calcium polysulfide potential to control the disease and its effect on the phyllosphere-fungal diversity. A positive correlation was found between concentration of calcium polysulfide and inhibition of fungal growth in the media. Otherwise, a negative correlation was found between concentration of calcium polysulfide and fungal biomass. Disease severity of cocoa treated by calcium polysulfide 80 mL L-1 every week was 11.67% significantly lower than control, i.e. 68.33%. Furthermore, it was also found that the index value of fungal diversity on the phyllosphere decreased by increasing concentration and interval of calcium polysulfide application.

ish color. The advanced infection stage, cocoa beans wrinkled and color changed to black and mummified [5]. This condition cause yield loss and poor seeds quality [6].
Calcium polysulfide is one of the potential control for pod rot plant disease. Although classified as synthetic inorganic fungicide and possibly affect the microflora on cocoa surface, but calcium polysulfide is allowed in organic farming and relatively safe to the environment [7][8][9][10][11][12]. Calcium polysulfide is capable to kill spores by disrupting the electron transport [13] as well as penetrating the fungal mycelium so that the fungus dies [14]. Affects the respiratory complexes of mitochondria by disrupting the flow of electrons in the respiratory chain, causing multi-site toxicity on a broad spectrum [15,16]. Affects enzymes in mitochondria [17]. Calcium polysulfide toxicity against non-target organisms is between non-toxic to moderate toxicity [18][19][20][21]. The purpose of this study were to evaluate the calcium polysulfide potential to control the disease and its effect on the phyllosphere-fungal diversity.

Relative Growth Inhibition and P. palmivora Biomass
Colonies diameter of P. palmivora for each replication was calculated from four quadrants, i.e. two transverse diameters and two longitudinal diameters. The influence of treatment on pathogen growth is showed in (%) value of relative growth inhibition (RGI) against control [22]: Biomass observation of P. palmivora, colonies on the surface of V8JA medium were washed with 10 mL HCl 4%. Filtered by No. 1 Whatman filter paper (filter paper weight has been known previously) and rinsed three times. Then dried in an oven with 70°C temperature for 3 days. After that the dry weight of P. palmivora was calculated from the dry weight difference between the empty filter paper and the filter paper contain P. palmivora.

In Vivo
The experiments were prepared based on factorial randomized block design, with 4 treatments and 3 replications. Each replication consists of 20 sample units (cocoa pod). Based on in-vitro experiment, obtained the best concentration of calcium polysulfide is 40 and 80 mL L -1 which used as in-vivo calcium polysulfide concentrations.
The first tested factor is concentration, consist of four levels: C: Control is sprayed with water; CP40: 40 mL L -1 of calcium polysulfide; CP80: 80 mL L -1 of calcium polysulfide; M2: 2 g L -1 of mancozeb. The second factor is interval applications, which consists of three factors: once in a week, once in two weeks and once in four weeks.

Level of Eficacy
Fungicide level of efficacy calculation conducted at last observation. Fungicide are effective when level of efficacy value > 50% and significantly different with control. Calculation using formula [24]: Description: Iac = Pathogen intensity of attack on control Iat = Pathogen intensity of attack on fungicide treatment

Fungal Diversity on the Phyllosphere
Cocoa pod sampling for fungal diversity were taken before application, 13 th week and 21 st week. To calculate fungal diversity using Shannon index formula (H') [25]. Species diversity index can be defined as high diversity if the value of H' > 3, medium diversity if 1 ≤ H' ≤ 3, low diversity if H' ˂ 1 [26].

′ = ∑ , ℎ =
Description: ni = the number of individuals (isolate) in each spesies N = the total number of isolates

RESULT AND DISCUSSION Relative Growth Inhibition (%)
Observation in two days after inoculation on 80 mL L -1 calcium polysulfide concentration was significantly different from 5, 10 and 20 mL L -1 calcium polysulfide concentrations and also the control (Fig. 2). Colonies diameter of P. palmivora on media treated with calcium polysulfide was smaller than control colony diameter. Likewise, enhancement of calcium polysulfide concentrations also causes decreasing P. palmivora colonies diameter ( Fig. 1 and Fig. 2). One level increase of calcium polysulfide concentration causes an increase in 5.978% relative growth inhibition y = -2.498 + 5.978x (Fig. 3).

Biomass of P. palmivora
Biomass is influenced by the fungi component such as mycelia that is formed. The colony diameter on calcium polysulfide treatment is smaller and thinner than the control. Similarly, comparison between calcium polysulfide concentrations also shows differences in P. palmivora diameter colonies and its thickness. The high calcium polysulfide concentration causes colony diameter become small and thin (Fig. 1). Its known that 80 mL L -1 calcium polysulfide treatment is significantly different from the other treatments and is not significantly different from 40 mL L -1 calcium polysulfide treatment (Table 2). One level increase of calcium polysulfide concentration causes a decrease in 0.004 g of biomass y = 0.031 -0.004x (Fig. 4).

Cocoa Pod Rot Attack Intensity
Observation and data analysis on cocoa pod rot (P. palmivora) intensity of attack was conducted every week until 21 st week. There is interaction between treatments (4 levels) with applications interval (3 levels). The interaction occurred in the 2 nd and 7 th -9 th week observation ( Table 3). This shows that the influence of each factor is not same for every other level of factor.
In general, observations at 7 th -9 th weeks of 80 mL L -1 calcium polysulfide treatment showed a significant effect on intensity of cocoa pod rot attack mean than other treatments at each level of applications interval. Calcium polysulfide 40 mL L -1 and M 2 g L -1 treatment in general at every level of application interval showed that average cocoa pod rot attack intensity was not significantly different except with 80 mL L -1 calcium polysulfide and control treatment. Once in a week application interval of 80 mL L -1 calcium polysulfide was found as the best treatment to control the disease.

Fungicide Level of Eficacy
Level of efficacy value at once in a week application interval of 80 mL L -1 calcium polysulfide is 70% (Table 4). This is effective for controling cocoa pod rot in-vivo, because efficication rate value more than 50% and significantly different compared with control.

Fungi Diversity on Cocoa Pod Surface
In general, the fungi diversity index has decreased, where the highest decrease occurred in once a week application interval of 80 mL L -1 calcium polysulfide treatment (Fig. 6). But from the third time cocoa pod sampling from the field, according to Fachrul [26] fungi diversity index is still categorized as medium (1.08-1.93).

Discussion
Calcium polysulfide has ability to inhibit growth, affect the biomass, affect the P. palmivora intensity of attack and affect the fungal diversity index. Because sulfur content in calcium polysulfide can inhibit fungal enzymes [27]. Sulfur affects fungal enzymes that can prevent mycelial growth [28]. Indicates the presence of polyphenols oxidation inhibition [27,29].
Calcium polysulfide is able to penetrate fungi mycelium so that the fungus dies [20]. Sulfur affects complex mitochondrial respiration by interrupting flow of electrons in respiratory chain, leading to multi-site toxicity [15,16,30].
Calcium polysulfide kills germinated spores [31]. This mode of action involves the interception of electrons in effective respiration chain by reducing element of sulfur to hydrogen sulfide, which is also toxic to cell proteins [16,32].
Sulfur as an effective inhibitor of conidia formation and germination and may affect multiple target sites in fungal cells. Among others, by inhibiting respiration by receiving electrons in the cytochrome c region that can interfere with proteins and bind heavy metals in cells [28,33,34].
At conidia, calcium polysulfide is able to prevent citrate production from exogenous acetate, and causes increased succinate, indicating enzymes blockage in pathway between acetate and citrate and inhibiting the succinoxidase system in Krebs cycle [35]. Sulfur as a hydrogen acceptor and provides interference in dehydrogenation reaction [34]. Free radicals from sulfur are formed in the sulfur-polysulfide-H2S conversion and these are highly reactive free radicals, which are toxic [35].

CONCLUSION
It was found a positive correlation between concentration of calcium polysulfide and inhibition of fungal growth in the media. Growth inhibition of fungi in the agar medium contained calcium polysulfide 80 and 40 mL L -1 was 17.921% and 11.295% respectively, significantly higher than control. It was found a negative correlation between concentration of calcium polysulfide and fungal biomass. Fungal biomass contained calcium polysulfide 80 and 40 mL L -1 was 1.5 mg and 8.1 mg respectively, significantly lower than control, i.e. 24.2 mg. Disease severity of cocoa treated by calcium polysulfide 80 mL L -1 every weeks was 11.67% significantly lower than control, i.e. 68.33%. Furthermore, it was also found that the index value of fungal diversity on the phyllosphere decrease by increasing concentration and interval of application calcium polysulfide.