Exploration on Soil Actinomycetes Against Phytophthora sp. Causing Root Rot of Cassava and Plant Growth Promoting Activities

MATERIALS AND METHODS

Soil sampling: Soil samples were collected from cassava fields of 10 different crop systems in tropical dry sandy in 3 provinces of Thailand, Lopburi, Nakhon Pathom and Kanjanaburi. The samples were kept at 4°C until use.

Isolation of soil actinomycetes and phytopathogenic fungus: Actinomyces were isolated from cassava field soil. Each sample was diluted to go on serial dilution and plate on humic acid vitamin agar (Hayakawa and Nonomura, 1987) supplemented with 50 mg L^–1 of cyclohexamide and 20 mg L^–1 of nalidixic acid. The isolation plates were incubated at 35±2°C for 7 days. The colonies had been transferred to International Streptomyces Project (ISP) medium No. 2 agar (Shirling and Gotlieb, 1966; Khucharoenphaisan and Sinma, 2011) plates for purity check.

Phytophthora sp. causing root rot of cassava was isolated from infected cassava. The isolated fungus was cultured on V8 agar at 35±2°C. Morphology under microscope showed mycelium with sporangium. It was identified as Phytophthora sp. using Internal Transcribed Spacer (ITS) region sequencing (Boonlue et al., 2003).

Screening of antifungal activity of actinomycetes: Ninety eight strains of soil actinomycetes were cultured on ISP medium No. 2 agar. The cultures of those actinomycetes were single streak on one side of PDA (potato dextrose agar) at distance of 1.0 cm from edge of plate and then incubated at 35±2°C for 7 days. The active mycelium of Phytophthora sp. was cut and transferred to the middle of cultured actinomycetes plate. The inhibition of mycelium growth was measured after 7-day cultivation. The inhibition was evaluated by radius of fungal colony compared to control culture. Three replications were done.

Test of culture filtrates: The effective soil actinomycetes were inoculated in ISP medium No. 2 broth and then incubated at 35±2°C for 7 days with shaking condition at 150 rpm. The cultured supernatants were filtrated using sterile cotton as supernatant. The supernatant with and without heat treatment at 100°C for 15 min were tested against phytopathogenic fungus of cassava activity using agar well method. The active mycelium of Phytophthora sp. was cut and transferred to the middle of cultured actinomycetes plate. The fungal plate was incubated at 35±2°C for 5 days. Thirty microliter of supernatant with and without heat treatment was put to each well and then inhibition of mycelium growth was measured. Three replications were done.

Identification of selected actinomycetes: The selected actinomycetes were tentative identified into genera based on morphological characteristics using crosshatch streak technique (Shirling and Gottlieb, 1966; Khucharoenphaisan et al., 2011). The morphological characteristics were observed as colony shape, color, sporulation, soluble pigment on ISP No. 2 and humic acid vitamin agar after 14-day cultivation at 35±2°C (Cuesta et al., 2012).

The 16S rDNA amplification of actinomycetes was prepared by PCR using universal primer 9F (5’-GAGTTTGATCC TGGCTCAG-3’) and 1541R (5’-AAGGAGGTGATCCAGCC-3’). The PCR products were purified and directly sequence using a Big Dye Terminator V3.1 cycle sequencing kit (Applied Biosystems) and the universal primers 9F (5’-GAGTTTGATCCTGGCTCAG-3’), 785F (5’-GGATTAGATACCCTGGTAGTC-3’), 802R (5’-TACCAGG GTATCTAATCC-3’) and 1541R (5’-AAGGAGGTGATCCAGCC-3’) (Khucharoenphaisan et al., 2012). The nucleotide sequences were compared with other bacteria using the Genetyx version 5.0. The phylogenetic tree was constructed by using the neighbor-joining method in MEGA version 4 software. The topology was evaluated by bootstrap analysis based on 1000 resamplings (Felsenstein, 1985).

Indole acetic acid (IAA) production: Selected soil actinomycetes was determined for its IAA production using colorimetric assay according to the methods of Goudjal et al. (2013) and Gangwar et al. (2012). The actinomycetes were grown on yeast malt extract broth containing 0.2% L-tryptophan and incubated at 35±2°C with shaking at 150 rpm for 5 days. Cultures were centrifuged 9000 rpm for 5 min. One milliliter of supernatant mixed with 2 mL of the Salkowski reagent. Absorbent was read at 530 nm using spectrophotometer. The level of IAA production was estimated by comparison with IAA standard.

Production of inoculums: The inoculum was prepared by placing 100 g of rice grain into plastic bag and autoclaving at 121°C for 15 min and then inoculated with S. malaysiensis or Phytophthora sp. and incubated at 35±2°C for 1 week after that mix with coconut dust in ratio 1:1. The sterile rice grain and coconut dust without inoculation were used as control using in the cassava fields experiment.

Fields experiment: The inoculum was dispersed in the soil after planting cassava for 2 month. In total, there were four treatment combinations: (T1) Control without microbial inoculation (T2) Inoculation with S. malaysiensis (T3) Inoculation with S. malaysiensis and Phytophthora sp. and (T4) Inoculation with Phytophthora sp. The efficiency of actinomyces was monitored by growth of the cassava. Twenty four cassavas per treatment were investigated.

Data analysis: The student t-test was used to determine significance fungal inhibition by supernatant and living cell of actinomycetes.

RESULTS AND DISCUSSION

Antifungal activity of actinomycetes: Among 98 isolates of actinomycetes from cassava field soils, 38 isolates showed antifungal activity against Phytophthora sp. as more than 60% (Fig. 1 and Table 1). All effective actinomyces were cultured in liquid medium and supernatants were collected for inhibition test.

Fig. 1(a-c):	Colony of Phytophthora sp. isolates from infected cassava as a (a) Control and (b, c) Antifungal activity of actinomycetes stains after incubated on PDA at 35±2°C for 7 days

Fig. 2(a-b):	Anti-fungal activity of actinomycetes culture supernatant (a) With and (b) Without heat treatment at 100oC for 15 min against Phytophthora sp.

Table 1:	Anti Phytophthora sp. activities of actinomycetes supernatant with and without heat treated at 100oC for 10 min

The result showed that supernatants from 16 strains had significantly inhibited the Phytophthora sp. grown on agar plate. However, there are only 6 isolates producing effective supernatants with heat resistant at 100^oC for 15 min (Fig. 2). This due to supernatant without heat treatment contained living cell of actinomycetes and it produced secondary metabolites or hydrolytic enzyme against the phytopathogenic fungus. Among these, isolate LB35 gave the highest inhibition of Phytophthora sp. in both supernatants with and without heat treatment. This indicated that isolate LB35 produced effective secondary metabolites with heat resistant such as bioactive compound to inhibit the phytopathogenic fungal growth. Some isolate of actinomyces had effective against the phytopathogenic fungal growth when use as cell or supernatant whereas supernatant with heat treatment were not. One of possible reason was the production of hydrolytic enzymes such as chitinase. Another one, the actinomycetes produced effective secondary metabolites to inhibit the phytopathogenic fungal growth but it not be heat resistant.

The finding agreed with the report of Suwan et al. (2012) that Streptomyces sp. isolated from soil that collected from Suthep-Pui National Park, Thailand have effective activity against phytopathogenic fungus. Moreover, Sinha et al. (2014) reported that Streptomyces plicatus was isolated from the soil could inhibit plant pathogenic fungi Phytophthora infestans and Sclerotium rolfsii to the tune of 80-100%. Elamvazhuthi and Subramanian (2013) also isolated Streptomyces from paddy soils and their antagonistic activity against Rhizoctonia solani, Helminthosporium oryzae, Curvularia lunata and Fusarium orysporum by using dual plate assay.

Production of indole-3-acetic acid (IAA): Among 6 isolates producing effective supernatant with heat resistant at 100°C for 15 min. Only strain LB35 could produce IAA with 50 μg mL^–1. Generally, IAA were effected to plant growth stimulating and controlling the mechanism of cell division, cell elongation and cell differentiation resulted to decrease of root length while increase number of root hair for more uptake of nutrient (Peciorek and Friml, 2006; Dobbelaere et al., 1999; Bennett et al., 1998). Various genera of Actinomycetes had ability to produce IAA such as Nocardia jiangxiensis and Actinomadura glauciflara, Aquilaria crassawa and it can be induce by the medium containing tryptophane (Sharma et al., 2013). However, Streptomyces malaysiensis has not been reported.

Molecular identification: The 16S rDNA sequence was generated for strain LB35 (1416 nucleotides). The phylogenetic tree of species Streptomyces constructed by Neighbor-joining method from MEGA4 program. The tree is rooted by the nucleotide sequence of Bacillus subtilis AE4-3. Scale bar shown distance values under the tree means 0.02 substitutions per nucleotide position. Bootstrap analyses were performed with 1000 re-samplings and percent values (>50) are shown at the branching points. Comparison of this nucleotide sequence with members of actinomycetes clearly showed that this strain belong to the genus Streptomyces (Fig. 3). The close relationship to Streptomyces malaysiensis is supported by both of treeing algorithms and a high bootstrap value (Fig. 3).

Application of strain LB35 on cassava field: The cultures of S. malaysiensis LB35 and Phytophthora sp. was applied on the 2 month growth cassava in the field, except negative control. The effect of Phytophthora sp. and S. malaysiensis LB35 on cassava growth on the field were checked every 2 months. After 4 month of planting (Fig. 4), treatment 1 (T1) Phytophthora sp. and S. malaysiensis LB35 were not added to cassava field. The cassava had height of 131.29±8.27 cm (Table 2). Addition only S. malaysiensis LB35 to cassava field, the cassava showed the highest height of 143.92±10.42 cm as shown on treatment 2 (T2). After added Phytophthora sp. and S. malaysiensis LB35 to cassava field (treatment 3, T3), the growth of cassava in term of height was slow decrease at 136.21±6.98 cm. In treatment 4 (T4) which inoculated only Phytophthora sp. in the cassava field revealed the growth similar to control experiment (T1). This indicated that S. malaysiensis LB35 can be use to stimulate growth of cassava because it produced IAA and also produce biological agent to inhibit Phytophthora sp. phytopathogenic fungus that cause of root rot.

Streptomyces malaysiensis has not been reported to be phytopathogenic microorganisms. Furthermore, it can produce bioactive compound to inhibit phytopathogenic fungus. Li et al. (2008) reported that S. malaysiensis produced malayamycin against Stagonospora nodorum causing of stagonospora nodorum blotch of wheat.

Table 2:	Growth of cassava after planting with Streptomyces malaysiensis LB35 and Phytophthora sp. on the field at 4 month

Fig. 3:	Phylogenetic tree of nucleotide sequence analysis of 16S rDNA of strain LB35

Fig. 4(a-c):	Cassava growth in the fields (a) After 2-month and (b, c) 4-month planting

Nonoh et al. (2010) isolated S. malaysiensis from national park soils in Kenya. It showed antagonistic activity to Fusarium oxysporum, Fusarium sp. and Colletotrichum kahawae. Streptomyces malaysiensis not only produced malayamycin, but also produced azalomycin F complex to inhibit phytopathogenic fungi such as Fusarium oxysporum, Rhizoctonia solani, Cladosporium cladosporioides, Fusarium chlamydosporum, Colletotrichum gloeosporioides (Cheng et al., 2010). Khucharoenphaisan et al. (2013) found that S. malaysiensis R58 produced thermostable bioactive compound on the culture broth against Colletotrichum gloeosporioides chi resulting to reduce chilli anthracnose.

CONCLUSION

The strain LB35 has high efficiency against phytopathogenic fungus Phytophthora sp. which isolated from infected cassava. This strain produced thermostable bioactive compound in the culture broth against Phytophthora sp. Moreover, it can produce IAA to stimulate growth of cassava in the field. Based on its 16S rDNA sequence, the actinomycetes strain LB35 is belonging to Streptomyces malaysiensis. Further study needs to be determined cassava production in the field. Moreover, it needed more potential bioactive antifungal compound and stimulating agents from S. malaysiensis, which are easily available in the soils.

ACKNOWLEDGMENTS

The authors thank to Dean of Faculty of Science and Technology for laboratory facilities, Phranakhon Rajabhat University. This research was supported by Higher Education Research Promotion (HERP). Under the office of the Higher Education Commission.