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 southern Thailand. part of Thailand. Five grams of the soil was diluted (10-4- 123 J Plant Dis Prot (2017) 124:41–50 43 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.