The Effect of Various pH Medium on the Secondary Metabollites Production from Trichoderma harzianum T10 to Control Damping Off on Cucumber Seedlings

Cucumber (Cucumis sativus L.) is one vegetable that has been widely consumed in Indonesia. Cucumber cultivation in Indonesia may decrease production due to a number of obstacles, including environmental mismatches, improper cultivation technology, and pests and pathogens existence. This occurrence affected low cucumber growth and production. One of the important diseases in cucumber plants is the seedling disease caused by Pythium sp. Some efforts to control the Pythium sp. have been conducted (Halo, et al., 2019). However, the utilizing of biological agents is considered to be the most effective and environmentally friendly. According to Kamala & Indira (2011), biological agents' secondary metabolites, such as Trichoderma spp. can be used to control the Pythium sp. It is well-known that Trichoderma spp. is antagonistic fungi for several pathogens (Naher, et al., 2017; Suada, 2017). One of Trichoderma species which widely used is T. harzianum. This fungal antagonist produces a secondary metabolite with the potential ability to suppress plant pathogens' development (Li, et al., 2019). Generally, the biosynthesis of the secondary metabolites of mycoparasitic fungi is influenced by the pH condition (Speckbacher & Zeilinger, 2018). Based on these reasons, it is necessary to observe the T. harzianum medium's appropriate pH to obtain the highest secondary metabolites production. This study aims to determine (1) the most suitable pH of the medium for T. harzianum T10 secondary metabolites production, (2) the effect of T. harzianum T10 secondary metabolites application to control damping-off disease, and (3) the effect of T. harzianum T10 secondary metabolites application to cucumber plant growth.


INTRODUCTION
Cucumber (Cucumis sativus L.) is one vegetable that has been widely consumed in Indonesia. Cucumber cultivation in Indonesia may decrease production due to a number of obstacles, including environmental mismatches, improper cultivation technology, and pests and pathogens existence. This occurrence affected low cucumber growth and production. One of the important diseases in cucumber plants is the seedling disease caused by Pythium sp.
Some efforts to control the Pythium sp. have been conducted (Halo, et al., 2019). However, the utilizing of biological agents is considered to be the most effective and environmentally friendly. According to Kamala & Indira (2011), biological agents' secondary metabolites, such as Trichoderma spp. can be used to control the Pythium sp. It is well-known that Trichoderma spp. is antagonistic fungi for several pathogens (Naher, et al., 2017;Suada, 2017). One of Trichoderma species which widely used is T. harzianum. This fungal antagonist produces a secondary metabolite with the potential ability to suppress plant pathogens' development (Li, et al., 2019). Generally, the biosynthesis of the secondary metabolites of mycoparasitic fungi is influenced by the pH condition (Speckbacher & Zeilinger, 2018). Based on these reasons, it is necessary to observe the T. harzianum medium's appropriate pH to obtain the highest secondary metabolites production.
This study aims to determine (1) the most suitable pH of the medium for T. harzianum T10 secondary metabolites production, (2) the effect of T. harzianum T10 secondary metabolites application to control damping-off disease, and (3) the effect of T. harzianum T10 secondary metabolites application to cucumber plant growth.

MATERIALS AND METHODS
The research was conducted from November 2018 to March 2019 in 2 stages, in vitro at the Plant Protection Laboratory and planta at screen house Faculty of Agriculture, Universitas Jenderal Soedirman, Purwokerto.

In-vitro Assay
Isolation of Pythium sp. Pythium sp. isolated by grows cucumber seeds in cow dung. Further, the parts of plants infected with Pythium sp. were isolated. Preparation of T. harzianum. The antagonist used is a fungus of T. harzianum T10 obtained from the collection

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of Plant Protection Laboratory. The fungus was cultured by using aseptically in Laminar Air Flow (LAF), incubated for seven days. Secondary Metabolites Preparation. Secondary metabolites of T. harzianum were prepared in Potato Dextrose Broth (PDB) medium. The acidity levels were regulated according to the treatment (3-7) by adding 0.1 N HCL or NaOH. The 150 mL of PDB were inoculated with 3 cork drill of T. harzianum aseptically. Medium added by T. harzianum was shaken using an orbital shaker for seven days at 1500 rpm at room temperature. Conidium density. Conidium density was calculated using the formula as follow: Where: S = conidium density t = number of spores in haemocytometer counting box d = dilution rate N = number of haemocytometer calculated boxes 0.0025 = volume of spore suspension 10 -6 = constants Chitinase Enzym Assay. Chitinase assay was carried out qualitatively by using chitin agar medium. The chitin agar medium's composition consists of 2% colloidal chitin, 0.1% K2HPO4, 001% MgSO4.7. H2O, 3% NaCl, 0.7% (NH4) 2 SO4, 0.05% yeast extract, 2% agar, and 1 L distilled water. The presence of the chitinase enzyme is characterized by the formation of a clear zone in the medium. ß-1,3-glucanase Assay. The examination to determine ß-1,3-glucanase was conducted qualitatively by the agar diffusion method. The 0.3g agarose solution was dissolved in 29 ml of distilled water and added with 1 ml of a sub-glucan substrate. The presence of the ß-1,3-glucanase enzyme is characterized by the formation of a clear zone in the medium. Inhibition Ability. The inhibition ability was calculated using the formula as follow:

In-planta Assay
Germination growth examination. The examination was carried out by growing cucumber seeds that soaked in T. harzianum secondary metabolites treatment at various pH levels, then grown on a petri dish inserted with filter paper moistened with sterile water. The observation was made by measuring the growth of roots and canopies on germinated cucumber seeds. Observations were implemented on the 7th day after planting. Percent germination is calculated using the formula as below: DB= Pythium sp. inoculation in cucumber plants and the application of secondary metabolites T. harzianum T10. Pythium sp. inoculated by insert 3 cork drills of Pythium sp. culture on the soil media. Cucumber seeds are placed on top of soil media. The secondary metabolites of T. harzianum with a density of 10 -6 poured on cucumber seeds as much as 10 ml/plant every five days. Disease incidence. Disease incidence was calculated using the formula: KP= AUDPC. The AUDPC calculated using the formula as follow:

In Vitro Assay
Based on the study results, the density of conidium T. harzianum T10 in the treatment of several pH mediums showed a difference (Table 1). The highest conidium density was reached by pH medium 5 as control (K0). All secondary metabolites of T. harzianum T10 grown on different pH medium showed that the secondary metabolites of T. harzianum shaken for seven days could grow in a medium pH 3-7. Meanwhile, the suitable pH medium to optimize T. harzianum is pH 5-7 (Singh, et al., 2018). Various pH of secondary metabolites medium T. harzianum significantly affects inhibitory ability (Tabel 1). The highest inhibition was found in the treatment with medium pH5 (K0), followed by the medium with pH 5.5 (K5) with percentage inhibition as 76.8% and 75.6%. It suggested that the secondary metabolites of T. harzianum have the ability to inhibit the growth of Pythium sp. This growth inhibition is proposed as an antibiosis reaction by the secondary metabolites compound of T. harzianum. Microscopic observations showed a change in Pythium sp. hyphae structure (Figure 1). The structure distortion of pathogens hyphae were occurred due to antagonistic activity through an antibiosis mechanism (Naglot, et al., 2015).
As shown in Table 1, the germination data explained that all treatments showed a high percentage of germination. It is probably caused by the use of healthy seeds with good viability, which is certified with high standards from the seed producer. According to Soares, et al. (2019), cucumber seed germination interfered with seeds' health quality. In Table 2, the secondary metabolites medium with a pH between 4.5 to 7 could produce enzyme ß-1,3-glucanase. Robinson (2015) found that enzyme activity is influenced by pH. In the case of ß-1,3-glucanase, it is suspected that a high acidic pH will cause cell metabolism disrupted, and a low pH condition causes the enzyme to work improperly.   Table 2, the secondary metabolites medium pH T. harzianum T10 with range 4.5 to 7 could produce chitinase enzymes. Hamid, et al. (2014) explained that the chitinase enzyme's optimum activity lay on a pH range 5 to 7. Meanwhile, the stability of the chitinase enzyme is laid on a pH range between 4 and 8. Table 3 showed that all treatments had a longer root than the canopy. It is suspected that the secondary metabolites of T. harzianum can produce growth regulators, such as hormones that can stimulate plant growth. This is in accordance with the opinion of Haneefat, et al. (2012), that application of T. harzianum secondary metabolites can increase gibberellins in plant roots. According to Bidadi, et al. (2010), the increase of adventitious and primary root length is caused by hormones' influence. Gibberellin hormone will support the formation of proteolysis enzymes, which will release a tryptophan as a precursor of auxin. It implied that gibberellins would increase the content of auxin, which induces rooting. The less optimum role of auxin hormones is due to certain compounds produced from the hormone gibberellin, which can be inhibitory. Therefore, the role of auxin is being interrupted.

In-planta Assay
Research result in planta showed that the incubation period of T. harzianum secondary metabolites with various concentrations showed significant results compared with K treatment (Tabel 4). The treatments of various concentrations were able to lengthen the incubation period of Pythium sp. as 84.82% against control. The application of mancozeb (F) showed similar effects with other treatment methods toward the incubation period (Tabel 4). Presumably, the active ingredient of mancozeb can suppress the infections of a pathogen. Therefore, the growth of pathogens is inhibited. This statement is in accordance with Gullino, et al. (2010), the mancozeb is a broad-spectrum fungicide that appropriates to control the fungal pathogen.
The incubation periods of all treatments of T. harzianum secondary metabolites in various concentrations were 42 days after inoculation. It showed that all treatments could suppress an incubation period of up to 100%. This evidence is in accordance with the opinion of Vinale, et al. (2014), a biological agent able to inhibit and control the pathogens by using secondary metabolites.
Based on the results, all treatments, including fungicide application, had a similar effect on disease incidence than control (Table 4). The activeness suspects the high disease incidence percentage in the control treatment of pathogens that are more adaptable and infectious to the plants. It is in accordance with Islam (2018), the occurrence of a disease is influenced by virulent pathogens, a conducive environment, and susceptible host plants. The interaction of those factors at the same time increases the development of the disease. The AUDPC is directly proportional to the disease incidence and incubation period (Figure 2). The control showed the highest disease incidence rate and the shortest incubation period. It is thought to be due to the absence of secondary metabolites of T. harzianum. Therefore, the suppression of the development of Pythium sp. does not occur. Meanwhile, T. harzianum secondary metabolites' application showed low AUDPC value, which was suggested as effective treatments to control Pythium sp.
The plant ages (days) Figure 2. The disease incidence of cucumber plants damping-off and AUDPC values.
As shown in Table 5, cucumber plants with T. harzianum secondary metabolites application had a longer root length than control (K). The shortest of cucumber plant root with control treatment is thought to be due to pathogen infection damage. Further, the infected root can inhibit plant growth and trigger plant death (Schroeder, et al., 2013). All applications of T. harzianum secondary metabolites were able to increase the root length in A1, A2, A3, B1, B2, and B3 treatment as 61. 58, 63.49, 62.95, 68.05, 67.7, and 56.57%, respectively. This occurrence revealed that T. harzianum secondary metabolites could produce enzymes to damage the cell wall of pathogenic fungi. Furthermore, it can inhibit the development of pathogens in plant tissues. Finally, the pathogens disable to infect plant roots. This occurrence is in accordance with the opinion of Vinale, et al. (2014). The secondary metabolites of T. harzianum are antibiotics, enzymes, and toxins that can inhibit pathogens in plant tissues with various mechanisms.
Based on the results as shown in Table 5, the application of secondary metabolites of T. harzianum was able to increase the fresh weight of plants in treatments A1, A2, A3, B1, B2, and B3 as 60. 25, 69.3, 66.48, 71.39, 70.52, and 70.32%, respectively. The occurrence was also identified by Ortuño, et al. (2017) in several plants such as lettuce, radish, and quinoa. The increase of biomass is suggested as an effect of cell division, expansion, and differentiation of fungal auxin-like compounds, besides improving plant nutrient uptake (Contreras-Cornejo, et al.. 2017).

CONCLUSIONS
1. The appropriate pH medium for the production of T. harzianum T10 secondary metabolites were pH 5 and 5.5. 2. Application of the T. harzianum T10 secondary metabolites on pH 5 and 5.5 with a concentration of 5, 10, and 15% could decrease the disease incidence. 3. Application the T. harzianum T10 secondary metabolites on pH 5 and 5.5 with a concentration of 5, 10, and 15% could increase crop height, the number of leaves, root lengths, and fresh crop weight.