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Antagonistic Activities of Endophytic Bacteria against Fusarium Wilt of Black Pepper (Piper nigrum).

Byline: Edkona Jenang Edward, Wong Sing King, Stephen Leong Chan Teck, Make Jiwan, Zakry Fitri Ab. Aziz, Franklin Ragai Kundat, Osumanu Haruna Ahmed and Nik Muhammad Ab. Majid

Abstract

Fusarium wilt is a fungal disease, which affects a broad range of plants including black pepper (Piper nigrum). Fusarium solani f. sp. piperis is a common causal agent of root rots and stem blight in black pepper. F. oxysporum Schl. f. sp. piperis, a less common but an important pathogen of black pepper. The biological approach to control F. oxysporum is becoming popular in many crop plants however there is lack of scientific record in black pepper. Endophytic bacteria were isolated from black pepper roots and cultured on nutrient agar. The bacterial isolates were screened for in vitro antagonistic activity against F. oxysporum through dual culture, mycelial growth, spore germination and double plate tests. Five isolates with promising antifungal activity were further identified through 16S rDNA sequencing. Isolates EB1 and EB2 showed highest antagonism against F. oxysporum mycelia with the percentage of inhibition up to 43 (Percent) and 41 (Percent) , respectively.

Isolated EB3, EB4 and EB5 produced clearing zones in spore germination test with radii measurements at 12.5-15.0 mm. The antifungal activities apparently involved the secretion of volatile and diffusible bioactive compounds. Analysis of the 16S rDNA sequences suggested the closest identities of the bacterial isolates as Bacillus megaterium, Bacillus cereus, Enterobacter sp. and Bacillus sp. Five endophytic bacteria isolates demonstrated significant control over both mycelia growth and spore germination of F. oxysporum. Some of these bacteria might possess additional beneficial plant growth promoting and insecticidal properties for the development of multi-function products in black pepper farming. (c) 2013 Friends Science Publishers

Keywords: Fusarium oxysporum; Biocontrol; Bacillus spp.; Enterobacter

Introduction

Black pepper (Piper nigrum L.), also known as the 'King of Spices', is one of the most widely used spices in the world. In addition, black pepper is also known as medicinal plant with several biological and medicinal properties such as antibacterial, antioxidant and antiulcer (Singh et al., 2004). Fusarium wilt is one of the major diseases, which causes extensive loss to black pepper plantation. The disease has been reported to shorten pepper vine life span from 20 years to 6-8 years and reduce productivity by half to 1.5 kg per plant (Anandaraj, 2000).

Fusarium solani f. sp. piperis is commonly regarded as the causal agent of root rot and stem blight in black pepper (Duarte and Archer, 2003). Nevertheless, a few records had linked the root and vascular infection of black pepper with F. oxysporum Schl. f. sp. piperis (Zhou and Chi, 1993; Sitepu and Mustika, 2000; Duarte, 2002).

Moreover, we have recently isolated a F. oxysporum from infected black pepper plant in the central region of Sarawak, Malaysia. Increased awareness in the deleterious effects of the chemical fungicides to human health, environment and natural ecosystems has led to a trend in developing biological means to control Fusarium diseases. Benchimol et al. (2000) had demonstrated significant protection of black pepper against F. solani f. sp. piperis by an endophytic bacterium, Methylobacterium radiotolerans. However, our literature search learned that there is lacking of scientific record about the biological control of F. oxysporum Schl. f. sp. piperis in black pepper. Numerous attempts have been taken to control other formae specials of F. oxysporum, which infect commercial crops such as banana (Mohammed et al., 2011), chickpea (Kaur et al.,2007), cumin (Haggag and Abo-Sedera, 2005), kidney bean (El-Mehalawy, 2004), strawberry (Nam et al., 2009) and tomato (Chandel et al., 2010).

In this study we screened through and isolated some endophytic bacteria from black pepper roots. The isolates were evaluated for biocontrol capability against the F. oxysporum using dual culture, mycelia growth, spore germination and double plate tests. The potential biocontrol candidates were further characterized for their antagonistic activities and identified through molecular means.

Materials and Methods

Root Sampling

Root samples of six-years-old black pepper plants were collected from three individual farms in Ulu Sarikei (2deg 7' 0" North, 111deg 31' 0" East), Sarikei Division, Sarawak, Malaysia. The samples were kept in clean and sterile containers, and brought to the laboratory within 24 h.Isolation of Endophytic Bacteria

Hundred g of the root samples were washed with sterile distilled water, surface sterilized by soaking in 70 (Percent) ethanol for 5 min, followed by 3 (Percent) sodium hypochlorite for 1 min, and rinsed with sterile distilled water. Parts of the roots were incubated on nutrient agar (NA) to confirm surface sterility. The roots were cut into short sections (2-3 cm) and macerated using mortar and pestle. One g of the macerated tissue was placed in a tube containing 9 mL sterile 0.9 (Percent) NaCl. The resulted suspension was subjected to ten-fold serial dilution and spread on NA plates in 3 replications. Incubation was done at room temperature (28 +- 2degC) for 3-5 days. Morphological characteristics of each isolate namely Gram reaction, shape and arrangement were recorded. Selected isolates were further identified based on their 16SrDNA sequences using the primers 16S7F (5'- AAGAGTTTGATCATGGCTCA-3') and 16S1542R (5'- TAAGGAGGTGATCCAACCGCAGGTTC-3').

DNA extraction was done by boiling bacterial cells in 2 (Percent) sodium dodecyl sulfate (SDS), followed by DNA precipitation in potassium acetate and absolute ethanol. PCR was performed in a thermal cycler using cycling conditions that consisted of an initial denaturation at 94degC for 3 min and then 35 cycles of denaturation at 94degC for 30 s, annealing at 45degC for 30 s, and extension at 72degC for 1.5 min. A final extension was performed at 72degC for 5 min.

Dual culture assay: Each bacterial isolates was assayed for antifungal activities against F. oxysporum isolated from infected black pepper plants based on the method described by Dikin et al. (2006). Fifteen isolates of endophytic bacteria from root samples were screened for their antagonistic activities. A 6-mm plug from the leading edge of a 9-days-old culture of each test fungus was placed at 1 cm from the edges of a 9-cm diameter petri dish containing potato dextrose agar (PDA). Streaks of endophytic bacteria were placed at approximately 1 cm on the edges at the other side of the plates. Negative control was not inoculated with any bacterial isolate. Bacteria that formed zones of inhibition (haloes without mycelial growth or distorted hyphae) over 2 cm were selected for further test. The barrier between the bacteria and the fungi indicated antagonist interaction between them. Cultures were incubated at room temperature and the percentage of inhibition of radial growth (PIRG) was recorded daily for 9 days.

The PIRG value was calculated as follows: Mycelial growth test: Plugs of mycelium were taken from the edge of six-days-old culture of F. oxysporum and dipped into broth cultures of the selected endophytic bacteria (109 cfu/mL) for 30 min and air dried in the lamina airflow chamber (Rahman et al., 2007). The mycelia plugs were placed at the center of PDA plates. Fungal plugs dipped in sterile distilled water served as negative control. The PDA plates were then incubated at room temperature. Each treatment was done in triplicate. The diameter of Fusarium colony was measured after 6 days of incubation. The percentage inhibition of diameter growth (PIDG) was calculated as follows: Spore germination test: Fifty (Mu)L of 105 cfu/mL suspension of F. oxysporum spores was spread over the PDA plate and allowed to dry for 10 min. Sterile paper discs (0.6 cm in diameter) containing 50 (Mu)L of nutrient broth (NB) supernatant of bacterial cultures (109 cfu/mL) were placed on the agar.

Discs received 50 (Mu)L of uninoculated NB served as controls. Radii of the clearing zones around the discs were measured after 24 h of incubation at room temperature (Palni et al., 2007).

Double plate assay: The endophytic bacteria and F. oxysporum cultures were first established respectively on NA and PDA plates for 2 and 5 days (Trivedi et al., 2008). PDA plate was inverted over the corresponding NA plate without cover lids, and the two plates were sealed together with parafilm strips. The plates were then incubated at room temperature for 3 days. For the control, the NA remained uninoculated. Growth inhibition of F. oxysporum by the endophytic bacteria was assessed based on the PIDG values.

Statistical Analysis

Data on dual culture assay, mycelia growth test, spore germination test and double plate assay were subjected to analysis of variance (ANOVA) while means were compared using Duncan's test at (p [?] 0.05) using Statistical Analysis System (SAS; Ver. 9.2; 2008).

Results and Discussion

Isolation of Endophytic Bacteria

Five (EB1-EB5) out of 30 bacterial isolates from the interior part of the pepper roots were selected for this study. These isolates were chosen based on rapid growth in a period of 24 h on NA media. Characterization of these endophytic bacteria (EB1-EB5) was carried out based on cell morphology (shape and arrangement), and Gram staining (Table 1). Four isolates (EB1, EB2, EB3 and EB5) were observed as bacilli, while only one (EB4) was coccus. The Gram staining results revealed that isolates EB1, EB2 and EB5 were Gram-positive bacteria, while isolates EB3 and EB4 were Gram-negative. According to Bergey's Manual of Determinative Bacteriology (Holt et al., 2000), genus Bacillus possesses rod-shaped and straight cells with cells measurements of 0.5-2.5 mm x 1.2-10 mm. The cells are often arranged in pairs or chains, with rounded or squared ends, which stain Gram positive and possess endospores that are oval or sometimes round or cylindrical (Holt et al.,2000).

Isolates EB1 and EB2 have morphological and Gram characteristics closely matched with Bacillus.

The primer pair, 16S7F and 16S1542R were designed based on the conserved regions of the bacterial 16S rRNA genes. The numbers on the primers indicate their positions in the gene based on the Escherichia coli sequence map (Baker et al., 2003). Hence, the amplified 16S rDNA was expected to be 1,536 bp in length which matched the sizes of PCR products of the bacterial isolates (Fig. 1). Based on the results obtained through the Basic Local Alignment Search Tool (BLAST) of the United States National Center for Biotechnology Information (NCBI; http://blast.ncbi.nlm.nih.gov/), partial 16S rDNA sequences of the EB1 and EB2 isolates revealed the closest match with the sequences of Bacillus megaterium and Bacillus cereus respectively (Table 2). Despite the highest 16S rDNA homology up to 97 (Percent) , EB3 displayed indistinct identities among Enterobacter cancerogenus, E. cloacae and E. hormaechei. Likewise, the identity of EB5 was ambiguity between two closely related bacteria, B. cereus and B. thuringiensis.

EB4 showed a short 16S rDNA sequence (237 bases) of only 81 (Percent) match with an uncultured bacterial clone. Thus, its identity remains unknown. It was not surprise to have three over five of our isolates from the genus Bacillus as earlier work had reported 30 (Percent) of the endophytic bacteria isolated from black pepper were Bacillus spp. (Aravind et al., 2009). Furthermore, Aravind et al. (2009; 2010) had identified B. megaterium and B. cereus among the endophytic bacterial isolates from black pepper.

germination tests revealed strong inhibition of F. oxysporum by EB3, EB4 and EB5 with clear zone radii measured 12.5-15.0 mm (Table 1; Fig. 4). Despite their high PIRG values, EB1 and EB2 only showed limited suppression on spore germination of 1.0 and 1.3 mm respectively. This contradictory phenomenon might be attributed to the nature of the bioactive compounds produced by the endophytic bacteria. When the dual culture tests done in PDA media, F. oxysporum received antagonistic effects from both non- volatile or diffusible and volatile compounds by the bacteria. The former were released and diffused through the agar, while the latter were emitted into the atmosphere in the petri dish. On the other hand, the inhibitory compounds in the broth media were most likely non-volatile owing to the low solubility of volatile organic compounds.

Therefore, the mode of action by EB1 and EB2 to control F. oxysporum was apparently through the production of volatile compounds as indicated by the PIDG values of double plate assay (Table 1). EB1 and EB2 belonged to the Bacillus genus, which comprises species such as B. subtilis and B. pumilus, which had been observed for producing inhibitory volatile compounds against F. oxysporum (Chaurasia et al.,1995; Kumar et al., 2008). In contrast, EB3, EB4 and EB5 appeared to suppress the fungal growth mainly via the diffusible bioactive compounds. As compared with the diffusible compounds, volatile compounds are relatively new research topic in microbial antagonism. Nevertheless, the latter compounds have gained increasing attention as they are perceived to be more powerful biological control mode. The volatile bioactive compounds could be delivered to a longer distanced target with a faster rate than the diffusible compounds.

It had been reported that the volatile compounds exhibited stronger antifungal activities than the diffusible compounds in rhizospheric strains of B. subtilis and Pseudomonas corrugate (Sood et al., 2007). However, in most of the endophytic antagonists (except for EB5), higher growth inhibition of F. oxysporum was observed for the combined effects of both volatile and non-volatile substances than volatile substances alone (Table 1). The percentage of inhibition in dual culture and mycelia growth tests were found slightly higher than the values of double plate assay. The differences were obviously attributed to the inhibitory substances produced by the endophyte isolates, which diffused through culture media, where both endophytic bacteria and F. oxysporum were grown in.

The isolate EB5 was unique for its highly inhibitory effect on spore germination of F. oxysporum (Fig. 4). EB5 showed close identity to B. cereus and B. thuringiensis (Table 2), and both species had been reported for the secretion of chitinases with antifungal activities against Fusarium (Chang et al., 2003; Reyes-Ramirez et al., 2004).

B. cereus is a common biocontrol agent against plant pathogens including F. oxysporum (Ahmed Idris et al.,2007). The biocontrol potential of B. cereus on F. oxysporum varies from weak to 57.7 (Percent) growth inhibition (Muhammad and Amusa, 2003; Ahmed Idris et al., 2007; Karkachi et al., 2010). This might be due to strain-specific antagonistic interaction between B. cereus and F. oxysporum. In this study, the B. cereus strain (EB2) exhibited 41 (Percent) suppression of Fusarium mycelia growth (Table 1). The antagonistic activity is comparable to earlier isolates by Ahmed Idris et al. (2007) with PIRG values range of 33.83 (Percent) -46.80 (Percent) . On the other hand, B. megaterium seemed to be a less popular bioantagonist. So far there were only two records on the control of F. oxysporum by B. megaterium (Ziedan and Farrag, 2002; Jung and Kim,2003). This is probably due to a much earlier contradictory report on the stimulative effect of B. megaterium on F. oxysporum (Naim and Hussein, 1958).

Nevertheless, EB1 isolate with closest identity to B. megaterium had demonstrated promising control activity with inhibition percentage of 43 (Percent) (Table 1) against F. oxysporum though this inhibition value was not as high as 62 (Percent) as reported by Ziedan and Farrag (2002). Jung and Kim (2003) had purified the antifungal antibiotic from B. megaterium strain KL39 and proven that it was active against a broad range of plant pathogens including F. oxysporum. The antagonist property of EB3 make its identity closer to E. cloacae as this particular species has been reported to cause 28.5-58 (Percent) growth inhibition of F. oxysporum f. sp. sesame and F. oxysporum f. sp. vicia (Abdel-Salam et al., 2007). The indistinguishable identity of EB5 was attributed to the lack of clear-cut difference between the 16S rDNA sequences of B. cereus and B. thuringiensis (Chen and Tsen, 2002).

Table 1: Morphological characteristics and antifungal activities of endophytic bacteria

Isolates###Cell Morphology###Gram Reaction###PIRG (Percent) Spore Germination###PIDG (Percent)

###Shape###Arrangement###(mm)###Mycelial Growth Double Plate

EB1###Bacillus###Chains###+###42.6 a###1.0 c###43.0 c###37.97 a

EB2###Bacillus###Pairs###+###41.0 a###1.3 c###41.1 e###34.86 a

EB3###Bacillus###Chains###-###25.8 b###12.5 b###29.8 d###24.00 b

EB4###Coccus###Clusters###-###25.0 b###13.0 b###27.7 c###22.20 b

EB5###Bacillus###Chains###+###24.2 b###15.0 a###24.8 b###2520 b

Notes: Mean values within a column followed by the same letters are not significantly different according to Duncan's test at (p Less than 0.05) PIRG = Percentage inhibition of radial growth; PIDG = Percentage inhibition of diameter growth

Table 2: Molecular identities based on 16S rDNA sequences

Isolate###Closest Match(es)###(Percent) Identity###Matched Bases###Accession No.

EB1###Bacillus nwgaterium (strain TSBF 723)###96###507/527###HM637290.1

EB2###B.cereus (strain WIF1 5)###95###858/906###HM480311.1

EB3###Enterobactercancerogenus (strain NB14 - 1A)###97###945/978###JN644583.1

###E.cloacae (strain G35-1)###HM217949.1

###Enterobacter hormaechei (subsp. steigerwaitli strain NM23 -1)###HM218110.1

EB4###Uncultured bacterium (clone KS4)###81###237/293###AF328160.1

EB5###B. cereus (strain HDDMGO3)###92###877/955###EU723819.1

###B. thuringiensis (strain W52618)###Z84586.1

Moreover, B. thuringiensis had been recorded for its antagonistic activity against F. oxysporum just like B. cereus (Knaak et al., 2007). Besides their antifungal activities, EB1 (B. megaterium), EB2 (B. cereus), EB3 (E. cloacae) and EB5 (B. cereus or B. thuringiensis) may possess additional desirable properties in promoting plant growth and health. Studies have revealed that B. megaterium (Chen et al.,2006) and B. cereus (Rylo Sona Janarthine et al., 2010) are capable of solublizing phosphate, while E. cloacae has long been shown for its nitrogen fixing ability (Zhu et al., 1986). Furthermore, B. thuringiensis is best known for its insecticidal activity which was widely used to control various insect pests in economic crops (Schnepf et al.,1998).

Conclusion

Five endophytic bacteria exhibiting potential in controlling F. oxysporum were isolated from black pepper roots. Based on the 16S rDNA sequences, two isolates were identified to species level as B. megaterium (EB1) and B. cereus (EB2), two were ambiguously identified at genus level as Enterobacter sp. (EB3) and Bacillus sp. (EB5), while one with identity remained unknown. Complement phenotypic characterization may be required to confirm the bacterial identities. On top of the antifungal properties, further exploration on the biofertilizer and bioinsecticide aspects of these endophytic bacteria may lead to the production of multi-function products for black pepper farming.

Acknowledgements

This MPB-UPM collaboration research project was fully funded by Malaysian Pepper Board (MPB).

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To cite this paper: Edward, E.J., W.S. King, S.L.C. Teck, M. Jiwan, Z.F.A. Aziz, F.R. Kundat, O.H. Ahmed and N.M.A. Majid, 2013. Antagonistic activities of endophytic bacteria against Fusarium wilt of black pepper (Piper nigrum). Int. J. Agric. Biol., 15: 291-296
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Author:Edward, Edkona Jenang; King, Wong Sing; Teck, Stephen Leong Chan; Jiwan, Make; Aziz, Zakry Fitri Ab.
Publication:International Journal of Agriculture and Biology
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Date:Apr 30, 2013
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