Fusarium Wilt Disease Control Using Biological Agents Trichoderma and Mycorrhizaeon Pepper

Fusarium oxysporum is the main fungus disease that can wither plants, especially pepper. The fungus spread through diseased soil or already withered plants and then infect plants from its roots. Doing control by using antagonistic fungi such as Trichoderma sp. and Arbuscularmycorrhizae have been widely performed. Trichoderma sp. is a fungus rich with an antifungal activity that produce metabolites, both volatile and non-volatile. These metabolites produced by Trichoderma can diffuse through the dialysis membrane which capable to slow several pathogens growth. Mycorrhizae creates mutual symbiosis between certain types of fungi with roots, also known as biological agents, capable to control F. oxysporum on pepper and to help antibiotics formation. The study was conducted at Laboratorium Hama dan Penyakit BPTP East Java, starting from January to May 2016. This study used completely randomized design (CRD) with treatment consisted of 16 combined doses of Mycorrhizae and Trichoderma, each repeated 4 times that produce 64 test units. Mycorrhizae dose used is 0.0; 1.0; 2.0 and 4.0 grams per polybag, while the Trichoderma dose used is 0.0; 15.0; 30.0; and 45.0 grams per polybag. Data were statistically analyzed by variance analysis and followed by a BNT test of 0.05. The results showed Mycorrhizae 4 g /polybag and Trichoderma 45 g /polybag application could increase the incubation period of F. Oxysporum fungus, shorten xylem discoloration and then reduce wilted plants percentage. Mycorrhizae application can boost Trichoderma fungus in order to reduce wilt disease found in pepper plants.


INTRODUCTION
Fusarium oxysporum is the main fungus disease that can wither plants, especially for pepper plants. The fungus spread through diseased soil or already withered plants and then infect other plants from its roots.
Mycelium is located around plant tissues and generally capable to isolate itself from diseased tissue or within the xylem vessels (Frank, 1972in Isnaini, et al. 2004). The disease can attack all stages of plants from early sprouting or even before to stage when plants start to flowering and fruiting. When young plants are infected, bottom stems become rotten to its wilted and shrink leaves to eventually die (Semangun, 1996).
F. oxysporum spread through already diseased soil which chlamydospore commonly found for then infect roots on pepper plants. This pathogen life cycle undergoes two phases, which are the phase of pathogenesis when the fungus live as a parasite on host plant and saprogenesis phase when fungus live as saprophytes that capable being the source of inoculum. This cycle can also cause disease on other plants and easily transmitted through wind, infected soil and groundwater as well as agricultural tools. (Doolite, et al., 1961in Winarsih, 1997.
Doing control by using antagonistic fungi such as Trichoderma sp. and Arbuscularmycorrhizae has been widely performed. Trichoderma sp. is a fungus rich with antifungal activity that produce metabolites, both volatile and non-volatile. These metabolites that produced by Trichoderma sp. can diffuse through the dialysis membrane which capable to slow several pathogens growth.
Mycorrhizae creates mutual symbiotic between certain types of fungi and roots (Budisma, 2014). This relationship produces very broad spectrum both in terms of host plants, fungi types, mechanisms in associating, effectiveness, microhabitat as well as diffusion.
Application of Mycorrhizae effectively increasing the nutrient uptake, heightening drought resistance, boosting growth hormone production and its regulator, giving protection from root pathogens and other toxic elements, while mushrooms get the benefit from photosynthetic supplies and evergreens. Mycorrhizae application can boost Trichoderma sp. in order to reduce wilt disease found in pepper.

MATERIALS and METHODS
Study was conducted at Laboratorium Hama dan Penyakit BPTP East Java, starting from January to May 2016. This study used completelyrandomized design (CRD) with treatment consisted of 16 combined doses of Mycorrhizae and Trichoderma, each repeated 4 times that produce 64 test units. Mycorrhizal dose used is 0.0; 1.0; 2.0 and 4.0 grams per polybag, while the Trichoderma dose used is 0.0; 15.0; 30.0; and 45.0 grams per polybag. Data were statistically analyzed by variance analysis and followed by a LSD test of 0.05.
Increasing F. oxysporum inoculum was performed in PDA media as well as increasing Trichoderma sp.on a medium-sized corn media grown in plastic bags. The seeds of pepper used are 1 month old or seeds that already passed 15 cm long. Trichoderma sp. and Mycorrhizae are given simultaneously with pepper seeds planted in poly bags according to respective dose and to each planting hole. Increasing F. oxysporum is performed 7 days after planting by immersing the pathogens into the soil with ratio 1 gram for 3 cm deep and then covered with transparent plastic to maintain moisture and to avoid contamination.

Mycorrhizae
and Trichoderma sp. application can greatly affect incubation period of F. oxysporum showed in Table 1  On the other hand, high dose of Mycorrhizae applied as single treatment resulted in slower F. oxysporum incubation period using4 g/polybag and the incubation period decreased to 17.07 days, while the incubation period of control treatment without Mycorrhizae was 12.59 days. Likewise, Trichoderma sp. application as single treatment with high dose of Trichoderma sp. also slowing the incubation period of F. oxysporum fungi using 45 g/polybag decreased the incubation period to 15.19 days while the incubation period of control treatment without Trichoderma sp. was 14 days. These events have occurred presumably because Mycorrhizae able to create hartique tissue that is difficult to be penetrated by pathogenic fungi, while Trichoderma sp. able to slow pathogenic fungi entry and movement. Agrios (1997) stated that environmental conditions that support plant growth and are less supportive for the development of pathogens will slow the incubation period and make pathogens need more time to infect plants (Figure 1 and 2). Application of Trichoderma sp. and Mycorrhizae also affect xylem discoloration length. Using both as single treatment resulted in slowing down the fungal infection at plant root done by F. oxysporum while Mycorrhizae work to suppress xylem discoloration length. Higher dosage of Trichoderma sp. and Mycorrhizae usage can shorten the length of discolored xylem and in some cases, there are even plants that grow without infected by F. oxysporum. Trichoderma sp. application with 45 g/polybag dosage resulted in shortest discolored xylem which was 3.69 cm, while control treatment without Trichoderma sp. resulted 12.46 cm. Another application of Mycorrhizae with 4 g/polybag dosage also resulted in shortest discolored xylem that was 3.92 cm, while control treatment without Mycorrhizae resulted 7.05 cm (Figures 3 and 4).
These events occurred presumably by Trichoderma sp. activities that are able to slow the development of F. oxysporum and Mycorrhizae that able to create an environment suitable for Trichoderma sp.  The lowest wilted percentage was shown by single treatment using Mycorrhizae at 4 g/polybag dosage with 44.68% while control treatment without Mycorrhizae resulted 59.41% (Figures 5 and 6). Likewise, single treatment using Trichoderma sp. with 45 g/polybag dosage resulted in 35.81% while control treatment without Trichoderma sp. is 77.59%. These events occurred because Mycorrhizae are able to increase the number of activity of antagonistic fungi inside soil yet able to reduce Fusarium activity in infecting plants. Cantoso, et al. (1997) stated that such conditions increase the competition between antagonists and pathogens inside soil causing pathogenic to lose. On the other hand, high dosage of Trichoderma sp. able to reduce disease progression more quickly and its chance to develop wider. Semangun (1996) stated that Fusarium fungi can infect through the tip of uninfected roots, where fungi develop in the parenchymal tissue and then settle to develop in the vascular bundles.   Papavizas (1985) stated that Trichoderma sp. capable to suppress the development of pathogens in several ways which are antagonist, parasitism and competition. Besides that, the environment can also affect the development of Mycorrhizae and Trichoderma sp. to suppress the rate of development of pathogenic fungi. The faster incubation period triggers faster F. oxysporum to infect the xylem vessels and resulted in longer discolored xylem. According to Semangun (1996) that Fusarium fungal infection starts from the roots, goes up to the base of the stem with white hyphae started to appear covering the stem skin, and continues to run to the top of the stem. Hyphae color will gradually changeto blackishor brownish when the infected area became greater and longer. By giving Trichoderma sp. as an antagonistic fungus for soil, it is expected to suppress the development of disease from inside soil. According to Sivan (et al 1987), Trichoderma sp. will develop primarily on the surface or the tip of the root so that it slows down contact and infection of the disease.

CONCLUSION
Application of Mycorrhizae 4 g/polybag and Trichoderma sp. 45 g/polybag are able to increase the incubation rate of fungus F. Oxysporum. Meanwhile, application of Mycorrhizae 4 g/polybag are able to reduce discoloration on Xylem by 44,68% rather than on control by 59,41%. On the other hand, application Trichoderma sp. 45 g/polybag are also able to reduce discoloration on Xylem by 35,81% rather than on control by 77,59%.