Biological Control 57 (2011) 236–245 Contents lists available at ScienceDirect Biological Control journal homepage: www.elsevier.com/locate/ybcon Isolation of endophytic endospore-forming bacteria from Theobroma cacao as potential biological control agents of cacao diseases ⇑ Rachel L. Melnick a, , Carmen Suárez b, Bryan A. Bailey c, Paul A. Backman a a The Pennsylvania State University Department of Plant Pathology, 207 Buckhout Lab, University Park, PA 16802, USA b Instituto Nacional Autónomo de Investigaciones Agropecuarias, Estacion Experimental Tropical, Km 5 vía Quevedo-El Empalme, cantón Quevedo, Casilla 24, Pichilingue, Provincia Los Rios, Ecuador c USDA-ARS Sustainable Perennial Crops Lab, 10300 Baltimore Ave., BARC-WEST BLDG 001 Room 223, Beltsville, MD 20705, USA article info abstract Article history: Sixty-nine endospore-forming bacterial endophytes consisting of 15 different species from five genera Received 15 December 2010 were isolated from leaves, pods, branches, and flower cushions of Theobroma cacao as potential biological Accepted 10 March 2011 control agents. Sixteen isolates had in vitro chitinase production. In antagonism studies against cacao Available online 14 March 2011 pathogens, 42% inhibited Moniliophthora roreri, 33% inhibited Moniliophthora perniciosa, and 49% inhib- ited Phytophthora capsici. Twenty-five percent of isolates inhibited the growth of both Moniliophthora Keywords: spp., while 22% of isolates inhibited the growth of all three pathogens. Isolates that were chitinolytic Theobroma cacao and tested negative on Bacillus cereus agar were tested with in planta studies. All 14 isolates colonized Bacillus the phyllosphere and internal leaf tissue when introduced with Silwet L-77, regardless of the tissue of Endophyte Cacao origin of the isolate. Eight isolates significantly inhibited P. capsici lesion formation (p = 0.05) in detached Moniliophthora leaf assays when compared to untreated control leaves. ARISA with bacilli specific primers amplified 21 Phytophthora OTUs in field grown cacao leaves, while eubacteria specific primers amplified 58 OTUs. ARISA analysis of ARISA treated leaves demonstrated that inundative application of a single bacterial species did not cause a long- term shift of native bacterial communities. This research illustrates the presence of endospore-forming bacterial endophytes in cacao trees, their potential as antagonists of cacao pathogens, and that cacao har- bors a range of bacterial endophytes. Published by Elsevier Inc. Introduction Additional research has demonstrated that the endophytic associ- ation of Trichoderma spp. activates the expression of cacao ex- Theobroma cacao L. is an economically significant crop, as the pressed sequence tags (ESTs) putatively related to defense genes seeds are processed into a range of cocoa products. Nearly 30% of (Bailey et al., 2006). the cacao crop is lost to disease annually (Hebbar, 2007). Although Despite the successes reported for fungal endophytes in sup- fungicide applications can improve yield, they are often too costly pressing cacao diseases, little is known about the presence or po- for small-holder farmers as well as pose risks to the health of the tential of bacterial endophytes. Work has demonstrated that applicator and the environment. For this reason, researchers have actinomycetes are common inhabitants of the cacao rhizosphere focused on altering the microbial ecology of the cacao tree to sup- (Barreto et al., 2008) and of pod surfaces (Macagnan et al., 2006). press diseases. Research has focused testing fungi as potential bio- Previous work by Melnick et al. (2008) demonstrated that an endo- logical control agents (BCAs). Rubini et al. (2005) demonstrated phytic Bacillus sp. originally isolated from tomato could endophy- that endophytic Gliocladium catenulatum reduced witches’ broom tically colonize cacao leaves for 60+ days and reduce severity of severity in small scale in planta studies. Arnold et al. (2003) dem- foliar Phytophthora disease in detached leaf assays. This research onstrated that cacao leaves inoculated with a consortium of seven further demonstrated that bacterial endophytes could be intro- endophytic fungal species had lower leaf mortality when duced into cacao, but the presence of native bacterial endophytes challenged with a Phytophthora sp. than endophyte-free leaves. in cacao and their potential for disease suppression still remained unknown. No published works on the cacao endophytes have utilized cul- ⇑ Corresponding author. Present address: USDA-ARS Sustainable Perennial Crops ture-independent methods to characterize the cacao beneficial or Lab, 10300 Baltimore Ave., BARC-WEST, BLDG 001, Room 310, Beltsville, MD 20705, pathogenic microbial communities, nor have researchers looked USA. Fax: +1 301 504 1998. at how application of endophytes impacts the native mutualistic E-mail addresses: [email protected] (R.L. Melnick), carmen.suarez@i- niap.gob.ec, [email protected] (C. Suárez), [email protected] (B.A. microbial communities associated with cacao. The current knowl- Bailey), [email protected] (P.A. Backman). edge of cacao microbial ecology is limited to isolates capable of 1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2011.03.005 R.L. Melnick et al. / Biological Control 57 (2011) 236–245 237 growing in a lab. Culture-independent technologies, such as auto- Tokyo, Japan) at the Penn State Genomics Core Facility. Sequences mated ribosomal RNA intergenic spacer analysis (ARISA), have from the forward and reverse primer were edited and aligned using been used in other woody perennials (Lambais et al., 2006) and Sequencher 4.7 (Gene Codes, Ann Arbor, MI), then the sequences could be useful in estimating the diversity of all bacteria present were compared to similar 16s rRNA sequences using Seqmatch of in cacao leaves. Additionally, this technology could be used to the Ribosomal Database Project (RDP 10.15, http://rdp.cme.msu.e- determine whether application of beneficial bacteria shifts the du/index.jsp)(Wang et al., 2007). Relatedness of isolates and re- populations of native microbes. lated bacteria (Ahmed et al., 2007; Anandaraj et al., 2009; Gilbert The objectives of this research were to obtain endospore-form- et al., 2004; Hoffmaster et al., 2004; Jeng et al., 2001; Narisawa ing bacterial endophytes from cacao trees, screen isolates as poten- et al., 2008) from the GenBank database was inferred using the tial biological control agents of cacao diseases, to determine neighbor-joining method (Saitou and Nei, 1987) of MEGA 4.0 (Ku- whether natural endophytes could persist in/on cacao leaves, and mar et al., 2008) to infer bacterial species. to determine whether endophytes were potential antagonists of cacao pathogens in planta. An additional objective was to utilize 2.3. Confirmation of heat tolerance by bacterial endophytes the culture-independent technology of ARISA to assess the bacte- rial community of cacao leaves and to determine whether applica- Confirmation of tolerance to heat (assumption of endospore for- tion of a single endophyte can shift the bacterial communities of mation) was conducted by growing bacteria in 800 ll of tryptic soy cacao leaves. broth (Difco, Franklin Lakes, NJ) in triplicate 1.7 ml vials. Tubes were incubated at 28 °C for 5 days, heated to 75 °C for 15 min, and then spread onto TSA plates. The plates were incubated at 2. Materials and methods room temperature (24 °C) for 24 h and observed for bacterial growth. The experiment was repeated to confirm results. 2.1. Isolation of endospore-forming bacterial endophytes from cacao tissue 2.4. Bacillus cereus assay Endophytic endospore-forming bacteria were isolated from sev- B. cereus HiVeg Agar (HiMedia Laboratories, Mumbai, India) was eral T. cacao ‘‘Nacional’’ clones that were previously identified as prepared following manufacturer’s directions to select B. cereus escaping cacao diseases on the Instituto Nacional Autonomo de group cells. Each isolate was spotted onto three replicate plates Investigaciones Agropecuarias Estacion Experimental Tropical (IN- which were incubated at 28 °C for 24–48 h and plates were ob- IAP-EET) in Pichilingue, Ecuador. Cacao tissue was collected and served for a colorimetric change (Mossel et al., 1967). B. cereus iso- sampled within 4 h. Four leaf disks (3.0 cm) were removed from lates BP24 and BT8 were used as positive controls. The experiment leaves, disinfected for 5 min in 20% bleach (Clorox), rinsed three was repeated to confirm results. times in sterile distilled water, triturated with a mortar and pestle in 3 ml 0.1 M potassium phosphate buffer (pH 7.0), were then 2.5. Chitinase assay transferred to a test tube, and incubated at 75 °C for 15 min. Fifty microliter of the suspension was plated onto tryptic soy agar Chitinase production was assessed by visualizing clearing (TSA, Difco, Franklin Lakes, NJ) and incubated for 48 h at 24 °C. around colonies on chitin nutrient agar (CNA) (Kokalis-Burelle Individual colonies were streaked on TSA. et al., 1992). Plates were incubated in the dark at 28 °C for 7– Pods were surface disinfected with 20% Clorox. The exocarp was 10 days. B. cereus isolates BP24 and BT8, known chitinase produc- aseptically removed and sections (0.42 cm3) of mesoderm were re- ers, were used for positive controls. The experiment was repeated moved. The periderm was aseptically removed from flower cush- to confirm results. ions and branches and internal sections (0.5 cm3) were sampled. Tissue was placed