Effectiveness of Liquid Trichoderma spp. Formulation as Biopesticides Against Phytophthora palmivora and Oncobasidium theobromae in Cocoa Plants ( Theobroma cocoa L.)

ABSTRACT

Trichoderma sp. has antifungal activity.In nature, Trichoderma is found in forest and agricultural soils or on woody substrates.Trichoderma was gained maximum attention as bio control agent due to the fact that it's effective against a huge number of soil-borne plant pathogenic funguses.There are two main methods on production of biopesticide that containing Trichoderma spp., either by solid state fermentation or by liquid state fermentation.Trichoderma sp., after being formulated as conidia mass, potentially could be used as biopesticide.This form of biopesticide will be produced by cell and spore propagation, until produce viable propagules and having high survival rate even after spread out on to the surface of target (leaf, plant etc.).This study was aimed to know the effectiveness of liquid Trichoderma spp.formulation as biopesticides against Phytophthora palmivora and Oncobasidium theobromae in Cocoa plants (Theobroma cocoa L.).The experiment was designed in a completely randomized design (CRD), by applying series of treatments consisting mix of extract of soybean (ES) and coconut water (CW).Tested treatments were: T0 (sterile distilled water 100% as control); T1 (100% ES); T2 (80% ES + 20% CW); T3 (60% ES + 40% CW); T4 (40% ES + 60% CW); T5 (20% ES + 80% CW); and T6 (100% CW).The results showed that the best treatment was T4 formula, which indicated by the highest number of spores density (9.76 × 10 8 ) during 48 hours incubation.Furthermore, this T4 formula treatment also gives the best effect as a biopesticide agent during application test in the cocoa pods.Biopesticides made from Trichoderma sp. are effective in controlling the pathogenic fungus Phytophthora palmivora that causes fruit rot and Oncobasidium theobromae that causes Vascular Streak Dieback (VSD) disease in cocoa plants, and more consequently the biopesticides are more effective in controlling Oncobasidium theobromae disease.The application of Trichoderma and its propagules as a biological control method can help reduce the use of pesticides and chemical fertilizers in agriculture in the future.

INTRODUCTION
Cocoa (Theobroma cocoa L.) is one of the most productive crops that contribute to increasing Indonesia's foreign exchange.Indonesia is currently the third major producer of cocoa after Ghana and Ivory Coast.This was a very important contribution to the Indonesian economy [1].One of the causes of low cocoa crop production is fruit rot disease caused by the fungus P. palmivora [2].P. palmivora causes serious losses to cocoa in Indonesia and around the world.This pathogen affects decrease the production of cocoa by 44% [3].
According to Hamdi and Lakani [4], the symptoms on infected cocoa pod caused by attack of P. palmivora fungus is brownish black skin around the fruit and fruit spotting finally happened overall.Infection with P. palmivora on cocoa pods directly through the skin tissue cocoa growing hyphae usually interselluler and forming Haustoria the host cells, or indirectly through the degradation of the cell wall of cocoa as an artificial wound.
In addition to P. palmivora, Oncobasidium theobromae also causes a reduction in fruit production as it causes Vascular Streak Dieback (VSD) disease.Because this pathogen attacks shoots and branches as well as xylem tissue, it causes the cocoa plant to die completely [5].This pathogen attack develops throughout the leaf surface which is brownish yellow in color and will eventually fall off so that it looks like rotting twigs [6].Diseased branches will split longitudinally when brown lines are visible in the xylem tissue [7].Furthermore, tissue death will spread to branches or to the main stem, so that the plant dies [8].
The control that has been done recently was mostly using chemical pesticide.The implementation of agricultural system prioritized more on the use of pesticide and chemical fertilizers, whereas by using such materials will only decrease the production temporarily.Beside, the negative impacts of the use of such materials is very substantial.It could lead to enviromental pollution and the death of other useful organisms [9].
Biopesticides are one method in place to reduce the impact caused by chemical pesticides [10].Natural pesticide or biopesticide is a material that is easy to decompose in the environment and environmentally friendly containing microbes as the active ingredient biopesticides such as fungi, bacteria or viruses that are antagonistic to other microbes or produce specific compounds that are toxic to both insects (pests) and nematodes (cause plant diseases) [11].One of the microbes used in the manufacture of a biopesticide is a fungus Trichoderma sp. because Trichoderma sp.antagonism against plant pathogens.
The results of previous studies reported that Trichoderma sp.fungi are able to control pathogenic fungi in plants such as Phytopthora sp.[12], and Fusarium oxysporum [13] and Phytophthora nicotianae [14].Trichoderma sp.controls pathogenic fungi in three ways, namely by antibiosis (clear zone as an inhibitor of pathogenic fungal growth), competition (fungi grow and take food and interfere with the growth of other fungi) [15], Trichoderma sp. also produces cellulase enzymes (degrading cellulose), chitinase (degrading chitin) [16], hemicellulase (degrading hemicellulose) [17] and glucanase [13] which can lyse the cell walls of plant pathogenic fungi [18].
Trichoderma sp. also functions as a decomposer, plant growth stimulator and as a biodecomposer, breaking down organic waste into quality compost [19].The proliferation of Trichoderma sp.fungi will occur if the fungus is in an environment that contains organic matter as a food source [15].Some organic wastes can be used as media for Trichoderma sp.propagation, such as coconut water and soybean extract.Coconut water is one of the abundant liquid organic wastes, in its utilization has not been maximized.Disposal of coconut water can cause acetic acid pollution, due to fermentation of coconut water waste, the acetic acid produced can affect soil acidity and have a negative effect on plants [20].Therefore, it can be used as a medium for making biopesticides, because it contains complete nutrients, namely 4% carbohydrates, 0.1% fat, 0.02% calcium, 0.01% phosphorus, iron and 0.5% total protein (9 g per L) [21].The carbohydrate content contained in coconut water is sucrose, glucose, fructose, mannitol, surbitol, and minositol [22], various hormones such as auxins and cytokinins.
Furthermore, soybean extract processing liquid waste contains dissolved organic matter which contains many protein compounds [23].Soybean extract liquid waste has been used in the manufacture of animal feed ingredients, but generally soybean extract processing waste is still disposed of without being reprocessed so that it has the potential to become a source of environmental pollution [24].
The role of Trichoderma sp.very large in suppressing fungal pathogen population, and able to live in wastes containing organic materials.Resistance of cocoa plants to fruit rot disease is commonly evaluated and tested by inoculating beneficial microorganisms such as Trichoderma sp.wowever, so far these methods have yielded inconsistent results.These research aims were: 1) Observation of symptoms of attacks by P. palmivora which causes fruit rot disease and O. theobromae which causes Vascular Streak Dieback on cocoa plants in the field, 2) Determine the effectiveness of biopesticides made from Trichoderma sp. which are propagated using basic substrates from soybean extract and coconut water and its application in P. palmivora (cause fruit rot on cocoa) and O. theobromae (causing vascular streak dieback).

Research design
This research was divided in two stages, namely: 1. Observation of symptoms of attacks by Phytophthora palmivora which causes fruit rot disease and Oncobasidium theobromae which causes Vascular Streak Dieback on cocoa plants in the field.
This research was conducted by direct observation to cocoa plantations in five villages, i.e ; Langaleso Village-Dolo Subdistrict, Sidondo I Village, Sigi Biromaru Sub-district, Petimbe Village, Makmur Village and Rahmat Village, Palolo Sub-District in Sigi Regency, Central Sulawesi, Indonesia.
Collection of pod and infected plants obtained from the results of observation and directly retrieval in local farm.Any pod affected by pod rot disease is caused by P. palmivora and VSD-infected plants caused by O. theobroma, sampling required for further analysis of the causing factors.
2. The second research stages was conducted in the Biotechnology Laboratory of Department of Biology, Faculty of Mathematic and Natural Sciences, Tadulako University.
The materials used in this study were PSA (Potato Sucrose Agar) media, distilled water, Trichoderma spp.inoculum (local isolate of Sigi Regency, Central Sulawesi), coconut water/CW (taken from fresh coconut fruits in the traditional market), soybean extract/SE (soybean water taken is liquid waste from the soybean industry), alcohol, and filter paper.O. theobromae fungus isolates were collected from infected cocoa plant twigs, P. palmivora isolates from infested cocoa fruits, sterile distilled water, tissue paper, sterile cotton, and methylene blue, and healthy cocoa fruits.
a. Preparation of Trichoderma sp.inoculums Pure culture of fungus Trichoderma sp., was propagated by culturing Trichoderma spp in a PSA medium in a petri, and then incubated at 30℃ for 5 days.Grown Trichoderma on the PSA, in a petri was harvested by adding 10 ml sterile distilled water, then the mycelium was stirred to separate the mycelium from the growth medium.Furthermore, stir until become formed suspense, then dilution spores (conidia) using haematocytometer and observation under a microscope.
b.The calculation of the density of spores (conidia)  Conidia calculated by using haemacytometer.
Haemacytometer used was NeubauerImproved type.
Which have large main box that divided into 25 squares and subdivided into 16 small boxes 1 small box with an area of 0.0025 mm 2 and a depth of 0.1 mm [25].
Conidia density was calculated of by taking 1 ml of spore suspension and placed slowly on the count area to fill the canal by using a micro pipette.Next put in stable position and closed with a cover glass.Calculating conidia density in the box count on 5 fields of view with a magnification of 100x. Culture of Trichoderma sp.aged 4 days was harvested, then filtered using a filter paper (dry weight known).
Filtering Using a funnel and erlenmeyer to obtain filtrate containing conidia.Conidia produced in the filtrate was calculated using a hemacytometer. According to Herlinda et al. [26], conidia density calculated using the method by Gabriel and Riyatno [27] as follows: Culture media used in this study was an extra soy in the form of liquid waste industries soybean oil and waste of coconut water.The culture is done in a culture bottle with media formula volume of 250ml per bottle.The composition of the treatment formula as follows: P0 (100% distilled water), P1 (100% ES); P2 (80% ES + 20% CW); P3 (60% ES + 40% CW); P4 (40% ES + 60% CW); P5 (20% ES + 80% CW); and P6 (100% CW).That formulated media according to treatment, was added to the culture bottles each of 250 ml.Media formula sterilized using an autoclave for 15 minutes at 121℃.Conidia suspension Trichoderma sp 2.5 ml inoculated on media formula with a concentration of 10 -6 conidia per ml -1 .The cultures were incubated at 30℃ shaker 150 rpm for 4 days.Furthermore, 0.5 ml biopesticides liquid formula that was spilled on cotton in the cocoa fruit.Then cotton covered with transparent tape.Next the fruit wrapped with tissue paper and transparent plastic, the development of P. palmivora were observed until day 7. Furthermore, the infected twigs O. theobromae dipped into the liquid formula biopesticides, dipped twigs placed in petri dishes with cotton in it.Each petri dish contains 5 pieces of twig or 5 spot.Four spot position within 2 cm from the edge of the Petri dish and 1 spot in the midst of a petri dish [28].All petridishes were incubated for 5×24 hours in a room temperature.
d. Data analysis Data were quantitatively analyzed variation (ANOVA), one-way ANOVA using the "Statistics Software Version 7".In the event of significant differences among treatments, then proceed to Test Multiple "Duncan".

Symptoms of attacks by Phytophthora palmivora
The symptoms of P. palmivora attack on cocoa pod can be seen in Figures 1 and 2. The results of observation of cocoa fruit attacked P. palmivora can be seen in Figures 1 and 2. Initial infection can occur through wounds, both wounds caused by mechanical friction between the fruit to one another, as well as small wounds by pest bites as well as large wounds hole of rat pest attack.Position of initial infection, can occur at the base of the fruit caused by the presence of a momentary puddle of zoospores can sprout and at the tip of the pod caused by hanging raindrops.P. palmivora is a water fungus that can stimulate the acceleration of zoospora release from sporangium, followed by zoospores sprout, then instantly invade pod tissue, after incubated 2-5 days can seen the symptoms of brown spots to blackish until finally decay cocoa pod as it knows as cocoa rot pod disease.Hyphae and sporangium of P. palmivora can be seen in the Figures 3 and  4.  P. palmivora attack on healthy pod and microscopic observation can be seen in Figure 3 and 4.
Raindrops can release the mycelium on the infected part of the pod and when accompanied by wind the loose spores will be spread and infections others pod.In addition to rain water droplets, the disease can also be spread through insects i.e.: ants and termites from the soil to cocoa pod.Ants can carry Phytophthora palmivora fungus for making a ground nest.This is in accordance with research [29].
Infected cocoa pod have symptoms of dark brown spots that can appear on the base, middle or end of pod.Infected cocoa pod collected from the plantation showed blackish brown spots.The P. palmivora spreading is caused by environmental humidity in the rainy season, high humidity helps the spores forming and increased infection in cocoa [30].Fungus in the ant species Iridomyrmex cordatus has the ability to carry many fungus P. palmivora, characterized by the appearance of black spots on the surface of the cocoa pod and P. palmivora can be isolated back in that pod.

Symptoms of attacks by Oncobasidium theobromae
The symptomps of O. theobromae attacks on cocoa plants can be seen in Figures 5 and 6.
Figure 6 shows that symptoms of O. theobromae attacks that appear are yellowing and drying leaves and bald ends of twigs.VSD can be caused by the lack of sanitation, because vector insects are commonly found in cocoa waste under the trees, too tight plant spacing and random trimming can incrase spreading of VSD.The branches contact between a healthy tree and a sick tree, or that too close trees faster the transmission, both by wind and friction directly.O. theobromae spread by the wind at night during high humidity.The presence of rain followed by moisture is more helpful during the disease spreading.Early symptoms attack on young leaves then spreading infection following xylem vessel tissue [31].High rainfall conducive to the pathogens development.
The leaves look yellow, drying and then fall off, leaving a bare twig, shown in Figure 4. O. theobromae develops in the xylem vein, obstructing the transport of water, nutrients and minerals from the root system to the leaf.Lacking nutrient in leaves, causing metabolic processes disturbed, eventually leaves begin yellowing, dry and fall off.The bare twig will dry out, and this happens systemically until the tree totally dead.
VSD attacks characterized by the first symptoms of yellowing leaves, especially on the first and second leaves, then the disease will enter into the xylem from the tip and base of twigs.Then the leaves will fall off from the branches followed by the drying of twigs and branches of plants [31].
Lacking of sanitation in cocoa farm will cause the pod rot disease causing by P. palmivora and Vascular Streak Dieback causing by O. theobromae [4].

The density of conidia of Trichoderma sp.
The results showed that the highest conidia density of Trichoderma sp on 4 days aftre incubation was achieved on the treatment media P4 (9.76 × 10 6 conidia ml -1 ) and it was significantly different with another treatments (P1, P2, P3, P5, and P6).While in the control treatment (P0), there was not found conidia (Figure 7).

The effectiveness of biopesticide to control and inhibit pathogen growth on cocoa pods
Effectiveness of biopesticide to control and inhibit pathogen P. palmivora on cocoa pods and Oncobasidium theobromae twigs cocoa can be seen in Figure 9.

Test fungal biopesticides in Phytoptohora palmivora
and Oncobasidium theobromae 1.The percentage of colonization of Trichoderma sp.Against Phytophthora palmivora Percentage of P. palmivora infection can be seen in Figure 10.
Figure 10 shows that the average percentage of infection with P. palmivora in treatment T1 (P.palmivora without biopesticides) is 90%, while seven another treatment P0, P2, P3, P4, P5, P6 and P7 treatment showed no colonization.Results in Figure 11 shows that the highest percentage of colonization of Trichoderma sp.against O. theobromae after 5 days of incubation was achieved by treatment P6 (100% CW) with an average of 0.93 or 93.33% and the lowest in the treatment P0 (control) with an average of 0%.
Trichoderma sp.produces three types of propagules that can be used as an ingredient in the formula controlling pathogenic fungi on plants consisting of hyphae,clamidiospora and conidia.Hyphae rarely used because hyphae not withstand the drying process.Klamidiospora initially thought to be used as a biopesticide active ingredient because of its ability to survive long enough period of time, but the capacity is very low germination.While conidia have walls thick and fast that the process of germination of conidia most developed in the mass production of a biological control agent [32].
The results showed that soybean industrial wastewater and coconut water have the potential as a growing medium for Trichoderma sp. to be used as a biopesticide.Conidia production in the P4 treatment (40% ES + 60% CW) of 9.76 × 10 6 ml -1 was higher than the 100% coconut water waste treatment of 4.56 × 10 8 per 100 ml and 100%, while in the treatment of 100% soybean industrial waste no conidia were found.This is confirmed by the results of Syahnen's [31], that the Trichoderma sp.fungus applied as much as 10 6 -10 8 conidia g -1 did not show any symptoms of P. palmivora infection which can be seen in Figure 3.The presence of biopesticides (Trichoderma sp.active ingredients) in cocoa tissue causes P. palmivora to be unable to colonize the tissue so that there are no visible symptoms of P. palmivora development infection.According Efendi et al [33], that Trichoderma sp.able to grow rapidly, compete, and produce antibiotics that broken host pathogen hyphae so as to inhibit the pathogen on the development of broad patches of cocoa fruit rot caused by the fungus P. palmivora.Furthermore, Sukorini et al. [34] stated that the suppression mechanism of Trichoderma sp.through mikoparasitisme which can inhibit the growth of pathogenic fungi with parasitism and cause lysis of the hyphae P. palmivora were able to remodel the cell walls of pathogens because the growth rate is faster than most pathogens.
The results showed that treatment P6 (100% CW) exhibited the highest Trichoderma sp.colonization, with an average percentage of 93.33% more effective against O. theobromae than the other treatments.Notably, treatment P0 (100% distilled water), used as the control, had the lowest effectiveness with an average colonization percentage of 0%.The more conidia contained in a liquid formula, the more hyphae biopesticides that will be generated to find food and a place to live so that there will be competition with mushrooms O. theobromae to get a place to live.This is in accordance with the opinion of Berlian et al. [35], that one way the fungus Trichoderma sp. to control fungal pathogens in plants is to compete for nutrients and a place to live.Once the fungus Trichoderma sp.get a life then Trichoderma sp.This will broken pathogenic fungi to take up the nutrients contained in the fungal pathogen [15].
The results of this research confirm the success of Trichoderma strains as biocontrol agents against pathogenic microorganisms, which are also expected to improve plant resistance, plant growth and development, leading to increased crop production and promising a clean environment to achieve sustainable agriculture in the future.

CONCLUSIONS
Based on the results, it can be concluded that the results showed that treatment P4 (40% soybean extract/ES + 60% coconut water/CW) can be used as a biopesticide because it can maintain and increase the growth of Trichoderma sp. until it reaches a density of 10 6 conidia ml -1 and produces high conidia, namely 9.76 × 10 6 conidia ml -1 .Furthermore, statistically, the treatment P6 (100% CW).showed the highest percentage of Trichoderma sp.colonization against O. theobromae colonization with an average percentage of 93.33% higher than the other treatments.Biopesticides made from Trichoderma sp. are effective in controlling P. palmivora, which causes cocoa pod rot, and O. theobromae, which causes Vascular Streak Dieback (VSD).This study confirms that Trichoderma sp. can replace the role of fungicides in controlling important cocoa diseases, maintaining soil health and sustainable agriculture.
/(n × 0.25) × 10 6 where, C = density of conidia per ml of solution t = total number of conidia in a box the observed sample n = number of samples box 0.25 = correction factor use box small scale samples hemocytometer c.Application of media formulation and Trichoderma sp.inoculums

Figure 1 .Figure 2 .
Figure 1.(a) P. palmivora attacking begins from the tip of the cocoa pod (b) P. palmivora attacking begins from the base of the cocoa pod (c) P. palmivora attacking begins from the middle of the cocoa pod

Figure 7 .
Figure 7.The conidia density of Trichoderma sp on various different media compositions3.4Morphology cocoa organ infected by fungi Phytoptohora palmivora and Oncobasidium theobromaeMorphology cocoa organ infected by fungi P. palmivora and O. theobromae can be seen in Figure8.