J Plant Dis Prot (2017) 124:41–50 DOI 10.1007/s41348-016-0051-y

ORIGINAL ARTICLE

Efficacy of indigenous Trichoderma harzianum in controlling Phytophthora leaf fall (Phytophthora palmivora) in Thai rubber trees

1 2 1 Athakorn Promwee • Punnawich Yenjit • Montree Issarakraisila • 1 3 Warin Intana • Chiradej Chamswarng

Received: 8 March 2016 / Accepted: 29 September 2016 / Published online: 8 November 2016 Ó Deutsche Phythomedizinische Gesellschaft 2016

Abstract Phytophthora leaf fall disease in rubber trees is compared with metalaxyl fungicide after inoculation with serious and is known to cause a reduction in both the P. palmivora for 120 days. quantity and quality of rubber latex in Thailand. This experiment was conducted to improve the effectiveness of Keywords Trichoderma isolate Á Rubber disease Á controlling rubber leaf fall disease in indigenous Tricho- Biological control Á Dual culture Á Bioactive compound derma harzianum. The results indicate that the T. harzia- num strain FR-NST-009, isolated from the rhizosphere soil of rubber trees, is more effective at inhibiting the mycelial Introduction growth of P. palmivora (66.22%) than the Thai commercial strain T. harzianum CB-Pin-01 (63.51%) with a dual cul- Para rubber (Hevea brasiliensis Muell. Arg.) is an eco- ture technique. This result is clear from the mycoparasitism nomically important rubber crop in Asia, and the leading of the T. harzianum strain FR-NST-009 under scanning producers are Malaysia, Indonesia, Thailand, India, Sri electron microscope. Moreover, the ethyl acetate crude Lanka and China. Southeast Asia supplies 92% of the extracts of the T. harzianum strain FR-NST-009 with a world’s natural rubber [1]. The southern part is the 500 lgml-1 concentration provided the most efficient majority for rubber plantation in Thailand. Phytophthora mycelial inhibition of P. palmivora by 92.42%. Four leaf fall disease is most prevalent in Hevea plantations in bioactive compounds isolated from crude extract and high rainfall regions and is severe where rainfall exceeds compound 3 provided the highest inhibition of sporangium 750 mm/month in June, July and August; leaf fall epi- germination by 98.83%. Compound 3 was then identified demics in Thailand occur during the long period of high as 6-n-pentyl-2H-pyran-2-one using nuclear magnetic res- rainfall, from June to December [2]. The disease is most onance spectroscopy. Under field conditions, the T. har- often caused by the fungus Phytophthora palmivora and zianum strains of RB-NST-028 and FR-NST-009 were able to some extent by a complex of other species, including P. to control leaf fall disease with no significant difference botryosa, P. meadii, P. citrophthora, P. nicotianae and P. capsici [3]. Three species of Phytophthora common in Thailand are P. palmivora Butler, P. botryosa Chee [4] & Warin Intana [email protected] and P. nicotinae var. parasitica [5]. The infection process of the soil-borne pathogen Phytophthora involving leaf 1 The Tropical Fruit Research Unit, School of Agricultural fall starts with the immature pod [2]andbecomes Technology, Walailak University, responsible for spreading the disease to mature leaves and Nakhon Si Thammarat 80160, Thailand 2 green stems [6]. Defoliation of 75% in a Para rubber tree Faculty of Agricultural Technology and Industrial may reduce the latex yield by up to 30–50% [7], as Technology, Nakhon Sawan Rajabhat University, Nakhon Sawan 60000, Thailand occurred in rubber trees of clone RRIM 600 in Thailand, which experienced a yield loss of 40% [8]. An obvious 3 Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Kamphaeng Saen Campus, sign of Phytophthora-induced leaf fall is coagulated latex Nakhon Pathom 73140, Thailand in a central petiole lesion and later dieback of terminal 123 42 J Plant Dis Prot (2017) 124:41–50 branches [4]. Moreover, zoospores of Phytophthora spe- fold dilution) with 45 ml of sterile water before 0.1 ml of cies are spread by rain splash from infected leaves onto this dilution was spread on Martin’s agar [26]. A pure the trapping panel, causing a black stripe with black lines culture of Trichoderma strains was identified as T. har- in the tapping cut and clogging the latex flow [7]. An zianum by the morphological and reproductive character- effective method that is recommended for controlling istics [27]. Phytophthora leaf fall is spraying with metalaxyl (25% Phytophthora spp. were isolated from symptomatic WP) or fosetyl-aluminium (80% WP) fungicides at con- rubber leaves of Phytophthora leaf fall using a tissue centration of 40 g/20 L water every 7 days [9]. However, transplanting technique [28]. The Phytophthora spp. were these synthetic fungicides have a harmful effect on human purified using single-spore isolation on a carrot agar (CA) health, leaving chemical residues in the environment, and were tested for pathogenicity by inoculating rubber impacting agricultural productivity and leading to fungi- leaves with the zoospore pathogen suspension at a con- cide resistance in several plant pathogenic fungi [10, 11]. centration of 1 9 104 zoospore ml-1. The Phytophthora Furthermore, synthetic fungicides are expensive due to a pathogen was identified as P. palmivora according to its tradedeficitinThailandin2012fromimporting6972tons morphological and reproductive characteristics, as descri- valued at 3883 million Baht [12]. Biological control bed by Sutton [29]. would therefore be an alternative method to avoiding these negative impacts in terms of cost, environmental Dual culture test and mycoparasitism concerns and health hazards. Trichoderma spp. are a biological control agent and All strains of T. harzianum were evaluated for their abili- have high efficacy in controlling numerous plant diseases ties to inhibit the mycelial growth of P. palmivora using a in many countries, such as the following: damping-off dual culture technique. Six-day-old T. harzianum on potato diseases in cucumber and Chinese kale [13, 14]; peanut dextrose agar (PDA) was cut with a sterile cork borer brown root rot [15]; chickpea wilt [16]; sugar beet leaf spot (3 mm diameter), and the agar plug of T. harzianum was [17]; strawberry black root rot [18]; avocado white root rot placed on one side of the CA Petri dishes (9-cm diameter). [19]; and berry fruit rot [20]. Trichoderma harzianum has Then, a 3-mm-diameter plug of 6-day-old P. palmivora been reported to be an effective biological control agent was placed on opposite sides of the CA Petri dish. The dual against Bipolaris oryzae [21]. Zhang et al. [22] found plates were incubated at room temperature (28 ± 2 °C) for treatment potential in T. harzianum against Sclerotinia 5 days. The experiment was conducted using a completely sclerotiorum, which causes stem rot disease in soy bean randomized design (CRD) with four replications. The per (Glycine max (L.) Merr.). Moreover, Kamala and Indira cent inhibition of mycelial growth was calculated using the

[23] found treatment potential in indigenous Trichoderma following formula: inhibition (%) = [(DC-DT)/ isolates and their liquid culture filtrates, which have high DC 9 100], where DC is the colony diameter of P. pal- efficacy against Pythium aphanidermatum, which causes mivora in the untreated control and DT is the colony damping-off disease of beans (Phaseolus vulgaris L.). The diameter of P. palmivora in the treatment [26]. The biological control of Trichoderma spp. may result from experiment was repeated twice. mycoparasitism, competition, antibiotics and induced The high efficacy strain of T. harzianum, which was able resistance in plant hosts [24, 25]. However, there has been to inhibit the mycelia of P. palmivora in an in vitro test, no research on the efficacy and mechanisms of indigenous was studied for the ability to parasitize the mycelia of the Trichoderma spp. in controlling Phytophthora leaf fall pathogen under a scanning electron microscope (SEM) disease in Thai rubber trees. (JEOL, JSM5600LV, England) according to the methods of Therefore, this research aimed to determine the efficacy Intana [26]. T. harzianum and P. palmivora were cultured of indigenous T. harzianum and their mechanisms in con- using a dual culture technique on CA. After the colony of trolling Phytophthora leaf fall in Thai rubber trees from P. palmivora was attacked by the mycelia of T. harzianum, Phytophthora palmivora. the activity zone was cut into small pieces (0.5 9 0.5 cm2), vapour-fixed with 2% (w/v) aqueous osmium tetroxide for 20 h at room temperature, air-dried and sputter-coated with Materials and methods gold palladium in a Palaron E 500 sputter coater (Nan- otech, Sempreps, England). The samples were then Trichoderma strains and the Phytophthora pathogen observed under SEM. This item was studied at the Scien- tific Equipment and Research Division at Kasetsart Trichoderma spp. were isolated from the soil on a rubber University Research and Development Institute, Bangkok, field in Nakhon Si Thammarat Province, in the . part of Thailand. Five grams of the soil was diluted (10-4-

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Inhibitory activity of crude extract Compound activity on spore germination

The crude extracts of the Trichoderma cultured medium The purified compounds were screened to inhibit the spore were filtered and tested for inhibitory activity of P. pal- germination of P. palmivora. The purified compounds were mivora mycelia growth. The agar plug of 7-day-old T. dissolved in 2% DMSO, diluted with sterile distilled water harzianum isolates were inoculated in 3-L Erlenmeyer and mixed with 0.1 ml of 1 9 104 sporangium ml-1 on flasks containing 1 L of 1/5 strength potato dextrose broth microplates for final concentrations of 100 and (PDB) and cultured with shaking at 100 rpm at 28 ± 2 °C 200 lgml-1. The experiment was arranged in a com- for 28 days. The cultures of T. harzianum were then filtered pletely randomized design (CRD), and each treatment through Whatman No. 1 and extracted with ethyl acetate consisted of three replicates. After incubation at 28 ± 2 °C

(CH3–COO–CH2–CH3). The crude extract was evaporated for 18 h, the sporangium of P. palmivora in each treatment to dry using a rotary vacuum evaporator and was stored at was stained with lactophenol cotton blue. The number of 4 °C. germinated sporangium was counted, and a total of 100 The crude extracts were tested for their ability to inhibit spores were measured in each of the three randomly the mycelia growth of P. palmivora, and the experiment selected fields under a dissecting microscope (magnifica- was conducted using a completely randomized design tion 4009). The per cent inhibition of sporangium germi- (CRD) with four replications. Each crude extract was dis- nation was calculated using the following formula: solved with 2% dimethyl sulfoxide (DMSO), mixed in inhibition (%) = [(the number of germinated sporangium PDB to a final concentration of 250 and 500 mg l-1. The in the untreated control (2% DMSO)–the number of ger- mixture of PDB crude extract was poured into a sterile minated sporangium in the treatment) 9 100]/the number plate. The mycelial discs of P. palmivora were then placed of germinated sporangium in the untreated control. at the centre of solidified agar plates and incubated at room temperature for 5 days. The colony diameter of P. palmi- Structural elucidation vora was measured, and its inhibition percentage of mycelia growth was calculated using the following for- The chemical structure of the compound was determined 1 mula: inhibition (%) = [(DC-DT)/DC 9 100], where DC is with H nuclear magnetic resonance (NMR) spectra the colony diameter of P. palmivora in the untreated con- recorded at 300 MHz and at 75 MHz in deutero-chloro- trol (2% DMSO) and DT is the colony diameter of P. form (CDCL3), on Bruker (Karlsruhe, Germany) spec- palmivora in the treatment [30, 31]. The experiment was trometers [32] and was compared with published data. repeated twice. Field experiment Compound studies A 6-month-old rubber tree clone RRIM 600 was trans- Isolation and purification of compound planted in a plastic pot (15 inch diameter) containing sterile soil (pH = 5.05, total N = 0.17%, P = 10.72 mg kg-1, The crude extract from T. harzianum FR-NST-009 was K = 62.88 mg kg-1) and sterile cow manure (total isolated using column chromatography containing silica N = 1.10%, total P = 0.48%, total K = 1.68%) at a ratio gel (SiO2). The crude extract was eluted with hexane of 1:5. Fifteen days after planting, 500 ml of the P. pal- 4 -1 (C6H14), followed by high polarity solutions of dichlor- mivora pathogen at 1 9 10 zoospores ml concentration, omethane (CH2Cl2), ethyl acetate (EtOAc) and methanol adjusted by haemocytometer, was then inoculated into the (MeOH), resulting in three fractions. These three fractions pot and incubated at 28 ± 2 °C for 7 days. T. harzianum at were separated by column chromatography containing 1 9 106 spore ml-1 concentration was added at 100 ml/ Sephadex LH-20 and eluted with methanol. The extract pot. The experiment was designed as CRD and as four was further purified by preparative TLC that was eluted replications per treatment. using a mixed solution of hexane and EtOAc in a ratio of

90:10. The TLC plate was sprayed with vanillin–H2SO4 to Disease severity observe each band of compounds. The compounds were then scraped from the plates, extracted with CH2Cl2 and Phytophthora leaf fall was observed after inoculation with dried under vacuum [32]. The purity of each compound P. palmivora for 120 days at 6° of severity (0–5) as fol- was verified on TLC plates using UV k max 254 nm, a lows: 0 = no visible symptom; 1 = 1–25% defoliation;

316-nm detector, vanillin–H2SO4 and an anisaldehyde 2 = 26–50% defoliation; 3 = 51–75% defoliation; standard spray reagent [33]. 4 = 76–100% defoliation; and 5 = dead tree [34]. The

123 44 J Plant Dis Prot (2017) 124:41–50 disease severity index (DSI) was then calculated using the Statistical analysis following formula: P ðÞScale  Amount of trees All data were subjected to an analysis of variance DSI ðÞ¼%  100 (ANOVA), followed by a comparison using the least sig- Maximum level  Total trees nificant difference (LSD). The significance level was set at P B 0.05. Trichoderma population

The population of T. harzianum was evaluated using the Results dilution plate technique after application of T. harzianum for 120 and 180 days. Ten grams of soil in the pot was Trichoderma strains and the Phytophthora pathogen added in a 250-ml flask containing 90 ml of sterile water and was mixed using a shaker at 120 rpm for 30 min. The The five Trichoderma strains that were isolated from the soil suspension was then diluted with sterile water at rhizosphere were T. harzianum according to their mor- -1 -5 10 -to10 -fold, and 0.1 ml of diluted soil solution phology and reproduction characteristics, as described by was dropped onto the surface of Martin’s medium with a Samuels [27], and they were indigenous to the southern micropipette and spread over the soil solution with a part of Thailand. For the Phytophthora strain isolated from sterile glass rod. The experiment was designed in CRD symptomatic rubber leaves during Phytophthora leaf fall with four replications per treatment. The number of T. disease, the virulent pathogen, in testing pathogenicity on harzianum was counted after incubation at 28 ± 2 °C for the leaf and stalk of RRIM 600 rubber tree, exhibited 100% 4 days [26]. disease incidence after inoculation at 28 ± 2 °C for 5 days. The Phytophthora strain had hyaline and non- Phytophthora population septate hypha and produced ovoid sporangium and chlamydospore, which was the same of P. palmivora. The population of P. palmivora in the soil of each pot was Moreover, its reproductive characteristic was also the same investigated after inoculation with P. palmivora for of P. palmivora as described by Sutton [29]. Therefore, this 120 days using a baiting technique. Five pieces of rubber Phytophthora strain was identified as P. palmivora. tree leaf (1 9 1cm2) were floated in the soil suspension (10 g soil/10 ml sterile water) in a Petri dish and incubated Dual culture test and mycoparasitism at 28 ± 2 °C for 2 days. The pieces of rubber tree leaf were then put on CA and incubated at 28 ± 2 °C for The results showed that all strains of T. harzianum effec- 2 days. The experiment was designed as a CRD with four tively inhibited mycelia of P. palmivora, at a range of replications per treatment. The percentage of colonized 51.35–66.22%, after incubation at 28 ± 2 for 5 days. T. leaves (PCL) was evaluated using the following formula: harzianum FR-NST-009 gave the highest percentage of LC/LT 9 100, whereas LC was the number of leaf pieces mycelial growth inhibition with 66.22%. The commercial colonized by P. palmivora and LT was the total leaf pieces strain of T. harzianum CB-Pin-01 provided lower inhibition [35]. by 63.51%. T. harzianum FR-NST-353, T. harzianum RB- NST-028 and T. harzianum RB-NST-003 had percentages Root colonization of Trichoderma of growth inhibition of 60.81, 58.11 and 51.35%, respec- tively (Table 1, Fig. 1). Root colonization of T. harzianum was observed after the The ability of T. harzianum to induce mycoparasitism on application of T. harzianum for 180 days. The rubber tree the mycelia of P. palmivora under SEM indicated that the roots were cut to a size of 1 cm in length, soaked in a T. harzianum strain FR-NST-009 could parasitize on the 0.53% solution of sodium hypochlorite for 5 min and mycelia of P. palmivora through colonization of Phy- washed with sterile water 3 times. The pieces of rubber tophthora hypha, drilling holes of Phytophthora hypha and root were then dried with sterile paper and put on a Mar- conidia reproduction on mycelia of P. palmivora (Fig. 2). tin’s medium. The experiment was designed in CRD with four replications per treatment. The colonization percent- Inhibitory activity of crude extracts age of rubber root was evaluated after incubation at 28 ± 2 °C for 4 days using the following formula: RC/ Antifungal activity against P. palmivora from the crude RT 9 100, where RC was the number of root pieces colo- extracts of T. harzianum cultures in a final concentration of -1 nized by T. harzianum and RT was the total root pieces 500 mg l was able to inhibit the mycelial growth of P. [26]. palmivora on CA to a range of 62.88–92.42% after 123 J Plant Dis Prot (2017) 124:41–50 45

Table 1 Effect of T. harzianum on mycelial growth inhibition of P. palmivora after incubation at 28 ± 2 °C for 5 days Treatments Place P. palmivora inhibition (%)

T. harzianum RB-NST-003 Rubber field soil from the 51.35c* T. harzianum RB-NST-028 Rubber field soil from the Thasala District 58.11b T. harzianum FR-NST-009 Forest soil from the Chang Klang District 66.22a T. harzianum FR-NST-353 Forest soil from the 60.81ab T. harzianum CB-Pin-01 Commercial strain from Thailand 63.51a P. palmivora Rubber leaf from clone RRIM 600 – * Mean values within the same columns followed by the same letter are not significantly different according to the Duncan’s multiple range test (P \ 0.05)

028, T. harzianum RB-NST-353 and T. harzianum RB- NST-003 provided 75.76, 67.42 and 62.88% growth inhi- bition, respectively (Table 2, Fig. 3).

Compound studies

Three fractions of ethyl acetate extract of the Trichoderma RB-NST-003 RB-NST-028 FR-NST-009 culture isolated using silica gel column chromatography were purified with Sephadex LH-20 column chromatogra- phy and preparative TLC. Four pure compounds were scraped from the TLC plates, including compound 1 (0.36 g), compound 2 (0.29 g), compound 3 (3.47 g) and compound 4 (0.33 g). All compounds were tested for an inhibitory effect on sporangium germination; compound 3 showed the highest FR-NST-353 CB-Pin-01 Control inhibition of sporangium germination at a concentration of 200 lg/ml by 98.83% (Table 3). 1H nuclear magnetic Fig. 1 Efficiency of T. harzianum to inhibit the mycelial growth of P. resonance (NMR) spectral data of compound 3 showed one palmivora on carrot agar after incubation for 5 days methyl proton at d 0.88, four methylene protons at d 1.30, 1.30, 1.62 and 2.49 and three vinyl proton at d 5.98, 6.19 13 incubation at 28 ± 2 °C for 5 days. Particularly, the T. and 7.21 (Fig. 4). The C NMR spectrum confirmed the harzianum strain FR-NST-009 induced the highest per- presence of two carbon–carbon double bonds at d 113.0 (C- 1 centage of mycelia growth inhibition at 92.42%. The 3), 143.8 (C-4), 102.7 (C-5) and 166.8 (C-6). These H and 13 commercial strain of T. harzianum CB-Pin-01 had lower CNMR spectral data were the characteristics of 6-n- inhibition by 76.52%. However, T. harzianum RB-NST- pentyl-2H-pyran-2-one as reported by Jeerapong et al. [36].

Fig. 2 Mycoparasitism of T. harzianum FR-NST-009 on P. palmivora, picture (a) shows Phytophthora normal hypha, and picture (b) shows that T. harzianum FR-NST-009 puncture hypha of P. palmivora and grow on

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Table 2 Effect of the crude extract of Trichoderma cultures on Field experiment mycelial growth inhibition of P. palmivora after incubation at 28 ± 2 °C for 5 days Disease severity Treatments P. palmivora inhibition (%) 250 lgml-1 500 lgml-1 The efficacy of T. harzianum in controlling Phytophthora leaf fall in rubber trees was tested under field conditions. T. harzianum RB-NST-003 31.25c* 62.88d The results indicated that 120 days after rubber trees were T. harzianum RB-NST-028 47.11b 75.76b inoculated with the inoculum of P. palmivora, all strains of T. harzianum FR-NST-009 65.12a 92.42a T. harzianum had high efficacy in controlling Phytophthora T. harzianum FR-NST-353 30.71c 67.42c leaf fall at a low disease severity index (0.00–11.67%), T. harzianum CB-Pin-01 49.51b 76.52b especially the T. harzianum strains FR-NST-009, RB-NST- P. palmivora ––028 and CB-Pin-01 (commercial strain), which presented a * Mean values within the same columns followed by the same letter disease severity index equal to a fungicide (metalaxyl), are not significantly different according to the Duncan’s multiple whereas the control 1 treatment inoculated with only P. range test (P \ 0.05) palmivora had the highest disease severity index, at 74.99% (Table 4).

Trichoderma population

The treatments of T. harzianum induced a population of 7.25 9 105–12.50 9 105 colony-forming units per gram soil (CFU g-1 soil) after application for 120 days and 2.25 9 105–9.50 9 105 CFU g-1 for 180 days, whereas RB-NST-003 RB-NST-028 FR-NST-009 the treatments with fungicide (metalaxyl), control 1 (with the pathogen) and control 2 (without pathogen) did not find Trichoderma species in the potting soil (Table 5).

Phytophthora population

After inoculation of P. palmivora for 120 days, the soil in each pot was evaluated for the number of P. palmivora using a baiting technique with rubber leaves. The treat- FR-NST-353 CB-Pin-01 Control (2%DMSO) ments of the T. harzianum strain FR-NST-009 had the Fig. 3 Efficiency of crude extracts from the Trichoderma culture in lowest of PCL, at 5%, followed by RB-NST-028 (10%), inhibiting the mycelia of P. palmivora on carrot agar for 5 days CB-Pin-01 (20%), FR-NST-353 (55%) and RB-NST-003 (65%). The treatments of T. harzianum had a significant per cent of colonized leaf discs (PCL) compared with a Table 3 Effect of compounds isolated from the crude extract of T. control 1 (with only pathogen), at 100% colonization harzianum FR-NST-009 culture to inhibit sporangium germination of P. palmivora (Table 4). Compounds Germination inhibition of P. palmivora sporangium (%) Trichoderma root colonization

-1 -1 100 lgml 200 lgml Root colonization of T. harzianum was determined after the 1 23.65b* 42.88b application of T. harzianum for 180 days. The treatments 2 26.71b 45.76b with T. harzianum strains RB-NST-028 and FR-NST-009 3 76.12a 98.83a had the highest root colonization percentage (100%) fol- 4 10.71c 32.42c lowed by FR-NST-353 (95%), CB-Pin-01 (95%) and RB- Metalaxyl 77.42a 98.74a NST-003 (85%), which had significant differences com- pared with control 1, control 2 and metalaxyl treatments, * Mean values within the same columns followed by the same letter which did not find Trichoderma strains colonized on the are not significantly different according to the Duncan’s multiple range test (P \ 0.05) roots (Table 5).

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Fig. 4 Structure (a) and the 1H NMR spectrum of 6-n-pentyl-2H-pyran-2-one (b) isolated from the crude extract of T. harzianum FR-NST-009

Table 4 Disease severity index of Phytophthora leaf fall (DSI) and plant diseases. The success of bioagents introduced into population of P. palmivora (PCL) under field conditions after inoc- soil causes physicochemical and biological factors that ulation for 120 days affect the establishment and serve as a repellent. The Treatments DSI (%) PCL (%) T. harzianum strains of RB-NST-003, RB-NST-028, FR- NST-009 and FR-NST-353 are indigenous in the southern T. harzianum RB-NST-003 11.67b* 65.00b part of Thailand, which is the area with the most Para T. harzianum RB-NST-028 0.00c 10.00cd rubber tree plantations. They were isolated from forest and T. harzianum FR-NST-009 0.00c 5.00d rubber soils in the southern part of Thailand and were T. harzianum FR-NST-353 5.00bc 55.00b tested for efficacy in inhibiting the mycelial growth of T. harzianum CB-Pin-01 0.00c 20.00c P. palmivora, and T. harzianum FR-NST-009 is more Metalaxyl 0.00c 0.00d effective in inhibiting the mycelial growth of P. palmivora Control 1 (only P. palmivora) 74.99a 100.00a than the Thai commercial strain T. harzianum CB-Pin-01 Control 2 (without P. palmivora) 0.00c 0.00d using a dual culture technique and provided the highest * Mean values within the same columns followed by the same letter percentage of mycelial growth inhibition in both the dual are not significantly different according to the Duncan’s multiple culture and crude extracts. Similarly, Kamala and Indira range test (P \ 0.05) [23] that found antagonistic potential of the indigenous Trichoderma isolates and their liquid culture filtrates that Discussion have high efficacy against P. aphanidermatum, which causes damping-off disease of beans. Biological control Phytophthora leaf fall disease is one of the most prevalent may result from direct or indirect interactions between a diseases in rubber plantations of Asia and is most often biological control agent and pathogen [37], such as caused by the fungus P. palmivora [3]. P. palmivora iso- mycoparasitism, and the synthesis of a hydrolytic enzyme, lated from symptomatic rubber leave was a virulent toxic compound or antibiotic, competition and induced pathogen, which caused 100% disease incidence on the resistance in a plant host [24, 25]. T. harzianum FR-NST- leaves and stalks of RRIM 600 after inoculation at 009 clearly indicated complete competition in a dual cul- 28 ± 2 °C for 5 days. Biological control is the best way to ture test and mycoparasitism under scanning electron avoid the negative or harmful effects on humans and the microscope, which T. harzianum FR-NST-009 colonized environment from chemical use; Trichoderma spp. are a and drilled holes in the hypha of P. palmivora, and conidia bioagent that has high efficacy in controlling numerous reproduction on mycelia of P. palmivora. Moreover, T.

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Table 5 Population of T. harzianum in the potting soil and rubber root colonization of T. harzianum (RCT) under field conditions Treatments Trichoderma population (CFU g-1 soil) Root colonization after application for 180 days (%) After application for 120 days After application for 180 days

T. harzianum RB-NST-003 12.50 9 105a* 2.25 9 105c 85.00a T. harzianum RB-NST-028 9.75 9 105a 2.50 9 105c 100.00a T. harzianum FR-NST-009 9.25 9 105a 5.50 9 105b 100.00a T. harzianum FR-NST-353 11.25 9 105a 2.75 9 105c 95.00a T. harzianum CB-Pin-01 7.25 9 105a 9.50 9 105a 95.00a Metalaxyl 0.00b 0.00d 0.00b Control 1 (only P. palmivora) 0.00b 0.00d 0.00b Control 2 (without P. palmivora) 0.00b 0.00d 0.00b * Mean values within the same columns followed by the same letter are not significantly different according to the Duncan’s multiple range test (P \ 0.05) harzianum FR-NST-009 clearly produced antifungal com- application. The overall of this research under field con- pounds in a crude extract test. Many repellent fungi pro- ditions indicated that the disease severity index of Phy- duce toxic compounds or antibiotic, and some antibiotics tophthora leaf fall disease of rubber was interrelated with have been shown to play a role in impeding spore germi- the population of P. palmivora in positive correlation and nation or killing cells [10, 24]. Our research found the correlated with Trichoderma population in soil and root antifungal compound of 6-n-pentyl-2H-pyran-2-one, which colonization in negative correlation. Nevertheless, the was identified by characteristics of 1H and 13CNMR indigenous strains of T. harzianum (FR-NST-009 and FR- spectral data, which is consistent with a report by Jeer- NST-028) have more a tendency to reduce population of P. apong et al. [36]. This compound was effective in palmivora and root colonization than the Thai commercial inhibiting sporangium germination of P. palmivora strain (T. harzianum CB-Pin-01), which indicated that the equivalent to metalaxyl fungicide. Among the volatile indigenous strains of T. harzianum isolated from soil of antifungal compounds produced by Trichoderma strains, southern part could adapt to environment as well. Addi- the most active and well documented is 6-n-pentyl-2H- tionally, induced resistance in plant by Trichoderma spp. is pyran-2-one (6-PAP), which is toxic to many common another mechanism for controlling plant diseases. plant pathogens, including Botrytis cinerea, F. oxysporum Nawrocka and Małolepsza [40] noted that Trichoderma f. sp. lycopersici, Fusarium verticillioides (moniliforme), strains could be to induce resistance both induced systemic Phytophthora megasperma, Rhizoctonia solani and Armil- resistance (ISR) and systemic acquire resistance (SAR) laria mellea [38]. pathways. On the other hand, this phenomenon depends on An experiment in open-field house conditions, T. har- Trichoderma strain, plant species, as well as biotic and zianum strains FR-NST-009, RB-NST-028, and CB-Pin-01 abiotic conditions. (Thai commercial strain) exhibited a disease severity index equivalent to metalaxyl fungicide. This result was due to competition, microparasitism and the antibiotic nature of Conclusion indigenous strains of T. harzianum that were verified by previously in vitro test. Moreover, this experiment indi- Trichoderma harzianum strains RB-NST-003, RB-NST- cated root colonization of T. harzianum strains to protect 028, FR-NST-009 and FR-NST-353 are indigenous in rubber from infection with P. palmivora. Harman et al. [39] southern Thailand. T. harzianum FR-NST-009 is effective reported that root colonization by the Trichoderma strain in inhibiting the mycelial growth of P. palmivora more results in increased levels of defence-related plant enzymes than the Thai commercial strain T. harzianum CB-Pin-01 in such as peroxidase, chitinase and b-1-3-glucanase. These both dual culture and crude extracts tests. T. harzianum mechanisms were the cause of a 0% disease severity index FR-NST-009 clearly exhibited mycoparasitism under and a reduction in the P. palmivora population in the scanning electron microscope. Moreover, T. harzianum treatments of T. harzianum strains FR-NST-009, RB-NST- FR-NST-009 clearly produced the antifungal compound of 028 and CB-Pin-01 in controlling Phytophthora leaf fall 6-n-pentyl-2H-pyran-2-one, which can be identified by the disease. Furthermore, the T. harzianum population (105 - characteristics of 1H and 13C NMR spectral data. This CFU g-1) indicated that it could survive in potting soil compound was effective in inhibiting sporangium germi- long enough to control the pathogen for 180 days after nation of P. palmivora equivalent to metalaxyl fungicide.

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