agronomy Study of Novel Endophytic Bacteria for Biocontrol of Black Pepper Root-knot Nematodes in the Central Highlands of Vietnam

: Black pepper is an industrial crop with high economic and export value. However, black pepper production in Vietnam has been seriously a ﬀ ected by the root-knot nematodes, Meloidogyne spp. The purpose of this study was to select active endophytic bacteria (EB) for the cost-e ﬀ ective and environmentally friendly management of Meloidogyne sp. Thirty-four EB strains were isolated. Of these, ﬁve isolates displayed the highest activity, demonstrating 100% mortality of J2 nematodes. These active EB were identiﬁed based on sequencing and phylogenetic analysis of the 16S rRNA gene; notably, all the potential endophytic bacterial strains belong to the genus of Bacillus. In greenhouse tests, Bacillus megaterium DS9 signiﬁcantly reduced nematodes in the soil and pepper plant roots with great inhibition values of 81.86% and 73.11%, respectively, with the lowest rate of nematodes built up at 0.23. This active antinematodes strain also showed good e ﬀ ect on promoting pepper plant growth. Some enzymatic activities, including chitinase and protease activity related to the biocontrol of Meiloidogyne sp., were also detected. The results investigated in the current study suggested that these selected EB strains may be good candidates for biocontrol agents of Meloidogyne sp., and plant promoting e ﬀ ects. The results also enhanced the novel active antinematode endophytic bacterial communities.


Introduction
Black peppercorn has long been considered a significant daily spice and among the most widely traded spices worldwide, amounting to 20% of all world spice imports [1,2]. This spicy plant has been cultivated widely in Vietnam, Indonesia, India, and Brazil. Of these, Vietnam is the largest producer and exporter of peppercorns with nearly 40% of the total 546,000 tons produced worldwide [3]. In Vietnam, black pepper trees have been mainly cultivated in the Central Highlands, the North Central Coast, Southeast Vietnam, and other southeastern areas. The Central Highlands and southeastern areas produce the largest amount of black peppercorns with about 124.5 hectares and production of 193.3 tons [4].

Isolation of Endophytic Bacteria
The black pepper plants roots were collected from healthy plants (3-7 years old) cultivated in Dak Nong province and kept in sterilized polyethylene bags. Endophytic bacteria were isolated according to the method described by Aravind [26]. The black pepper plant roots were washed with water, and then cut into pieces with the length of 1-2 cm. Tween 80 was used to treat the pieces for 10 min and sterilized water was then used for washing. Then 2% sodium hypochlorite solution was used for sterilizing the surface of the root samples for 10 min, and subsequently with 70% ethanol for 1 min. The samples were then washed six times with sterilized distilled water. The samples were mixed with phosphate buffer saline (PBS) pH 7·4, ground, and then centrifuged (600 rpm) at 5 • C for 2 min, and 100 µL of the suspension was inoculated into TSA medium and cultivated at 28 • C for 1-3 d. Single colonies were separated and sub-cultured on new TSA medium. All isolated strains were stored in 50% glycerol at −32 • C.

PCR Amplification, Sequencing, and Phylogenetic Analysis of the 16S rRNA Gene
The active bacterial strains were identified based on 16S rDNA gene sequencing. Genomic DNA from an overnight culture of each strain was extracted by the method described by Tran et al. 2018 [27]. The genomic DNA was used as a template for amplification by PCR. A nearly full-length segment of 16S rRNA gene nucleotides was amplified in a 100 µL reaction tube using the universal primers 27f (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 1492r (5 -TACGGYTACCTTGTTACGACTT-3 ). The 16S rRNA gene was amplified by iCycler thermal cycler (Bio-Rad, Hercules, CA, USA) using the following schedule: 94 • C for 5 min, repeated by 30 cycles of denaturation at 95 • C for 30 s, annealing at 55 • C for 30 s, and extension at 72 • C for 2 min. The amplified products were then separated by electrophoresis on agarose gel (1.5%, w/v). The target bands in the agarose gel were cut out and purified using a QIA quick PCR purification (Promega Co., Madison, WI, USA). Sequencing reactions were carried out in a CEQ8000 Genetic Analysis System (Beckman Coulter Inc., Brea, CA, USA) using a CEQ Dye Terminator Cycle Sequencing Kit (Beckman Coulter Inc., Brea, CA, USA).
The nucleotide sequences (from 1300 to 1440 bps) of the 16S rRNA genes were compared to known sequences in the DDBJ/Genbank/EMBL databases using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the taxonomic positions of the rhizobacteria isolates. The phylogenetic tree was made using Kimura's method and drawn using the Mega software version 6.0 after multiple alignments of the data by Clustal W [28,29].

Bacterial Strains Preparation
Bacterial strains were cultured on liquid medium containing beef extract (7 g), peptone (7 g), NaCl (50 g) in 1000 mL distilled water, and an initial pH 7.0, at a shaking speed of 150 rpm at 28 • C for 24 h. Thereafter, each endophytic bacterial strain was harvested via centrifuge at 8000 rpm for 10 min to remove the culture supernatant before the bacterial mass was suspended in saline buffer. Each strain was then arranged by spectrophotometry to 10 7 CFU mL −1 at a wavelength of 600 nm prior to the in vitro and greenhouse tests.

Anti-Nematode Activity In Vitro Assays
The nematicidal activity in vitro assay: 250 µL sterile distilled water containing around 30 nematode J2 of Meloidogyne sp. was mixed with 100 µL bac solution (10 8 CFU/mL) in a 96-well tissue culture plate. The mixture was kept at 28 • C for 24 h before the mobilized nematodes and immobilized J2 were counted. Nematodes that did not move when touched with a fine needle were defined as inactive (immobilized nematodes) [30] ( Figure 1C,D). The control experiment was conducted under exactly the same conditions as described above but without bacteria. All the testes were done with triplicates.
The effect of culture filtrates on nematode egg hatching: 100 µL culture filtrates were mixed with 250 µL sterile distilled water containing around 200 Meloidogyne sp. eggs in a 96-well tissue culture plate. The mixture was kept at 28 • C for three days; the hatched juveniles were then counted with the use of a low power stereoscopic microscope. The control experiment was conducted triplicates under exactly the same conditions as described above but 100 µL unfermented culture filtrate was used instead of fermented culture filtrates.

The Effect of Endophytic Bacteria on Reducing Nematodes and the Plant Promoting Effect for Black Pepper Trees in Plots Experiments
The most active anti-nematodes endophytic bacterial strains in the in vitro test were selected for the further bioassay in the greenhouse. Local Vinh Linh black pepper seedlings with five leaves were used for the plot experiments. The experiment was arranged according to the random distribution formula. Each experiment had six groups, including five experimental groups treated with selected bacterial strains and one control group (no bacteria treatment). Each group consisted of 30 pepper Agronomy 2019, 9, 714 5 of 12 seedlings, with one pepper seedling cultivated per pot. The distance between each group was 60 cm, with 50cm between each pot. The greenhouse conditions were set to 25-30 • C, 75-80% humidity, and a light intensity of 50-550 µmol m 2 /s −1 (measured from 08:00 to 16:00). Five-leaf-old seedlings were transplanted in plastic pots containing about 0.5 kg of sterilized soil, sand, and organic fertilizer in a ratio of 2:1:1 (v/v), respectively. Two times 100 mL of endophytic bacterial suspension with a density of 10 7 CFU mL −1 as inoculated into each pot two weeks apart. After two weeks, each pot was inoculated with 300 nematode J2. The control group received the same treatment as described above but saline was used instead of bacteria. Tap water was used for irrigation. Each of these treatments was used to fill ten plastic pots arranged in a randomized block design on benches in a greenhouse. After three months of endophytic bacterial inoculation, the black pepper seedlings were carefully removed and washed from the soil. Nematode parameters as numbers of J2 in 10 g of soil and in 1 g roots were counted. The percentages of efficacy and the rates of nematode build-up were calculated. The growth parameters, such as increased shoot length, increased root length, number of leaves, and chlorophyll a+b contents.

Enzymic Activity Assays
Protease activity of the culture supernatants (CS) of Bacillus megaterium DS9 was measured according to the techniques described in the previous report [31], with slight modifications. In brief, 0.1 mL of CS was mixed with 1 mL casein solution (1% in 50 mM phosphate buffer). The mixture was incubated at 37 • C for 60 min. Trichloroacetic acid (TCA) solution (5%) was added to stop the reaction before the mixture was centrifuged at 4000 rpm for 15 min. A total of 0.75 mL of this solution was then mixed with 1.5 mL of NaOH 0.28 N and 0.48 mL of Folin's reagent. It was left to rest for 15 min before being measured at OD 660 nm. Tyrosine was used as reference. One unit of enzyme activity was defined as the amount of enzyme needed to release 1 µmol of tyrosine per min.
The chitinase activity of CS was measured according to the methods described in our previous report [32]. In brief, 50 µL of CS, 100 µL of pNPg (p-nitrophenyl-N-acetyl-β-D-glucosaminide) (1 mg/mL) and 500 µL of sodium acetate buffer (50 mM, pH 5.8) were incubated at 37 • C for 30 min. A total of 350 µL of sodium carbonate buffer (50 mM, pH 10.7) was then added to the mixture before the final solution was measured at OD 415 nm. The chemical compound pNP (p-nitrophenol) was used as a reference. The amount of chitinase that produced 1 µM of pNP per min was defined as one unit of chitinase activity.

Statistical Analysis
For the statistical analysis of the results, the data on the experiments were subjected to analysis of variance (ANOVA), and means were separated (p ≤ 0.01) by Duncan's multiple-range test using Statistical Analysis Software (SAS-9.4, SAS Institute Taiwan Ltd, Taipei, Taiwan).

Isolation, Evaluation, and Identification of the Active Antinematodes Endophytic Bacteria in the In Vitro Test
Thirty-four endophytic bacterial strains were isolated from the 3-5 years black pepper roots in Dak Nong Province in the Central Highlands of Vietnam, and the antinematode effect was evaluated in the in vitro test ( Table 1). The results illustrate that almost all of the newly isolated endophytic bacterial strains showed potent activity. Seventeen strains displayed the potential antinematode activity with the mortality higher than 85.56%. Of these, five strains: DS5, DS8, DS9, DR2, and DR10 demonstrated the most efficacious antinematode activity with the mortality of 100%. The active strains were identified as Bacillus flexus DS5, Bacillus sp. DS8, Bacillus megaterium DS9, Bacillus sp. DR10, and Bacillus sp. DR2, based on the 16s rRNA gene sequences analysis. Phylogenetic analysis of the identified strains is illustrated in Figure 2, and the accession number of 16s rRNA genes of the selected endophytic bacterial strains is listed in Table 2.  The phylogenetic tree was made as per Kimura's method and created using Mega software version 6.0 after multiple alignments of the data by Clustal W. The numbers at the branches are bootstrap confidence percentages (%).  Table 3 show that all the five tested strains demonstrate reduced nematodes in soil and in pepper roots with inhibition values of 56.33-87.34% and 38.89-76.44%, respectively. The rates of nematodes built up in all the plots treated with RB were lower than that of the control plot. Of these tested strains, DS9 showed the most potential in biocontrol of nematodes with great inhibition values of 81.86% and 73.11%, both in soil and in pepper roots, respectively, indicating the lowest rate of nematodes built up of 0.23. In the greenhouse tests, all the strains showed no negative effects on the pepper plants. In addition, they also demonstrated a significant effect on plant growth promotion, including the increased shoot length (22.64-29.81 cm), formation of new leaves (3.79-4.19 leaves) and increased root length (6.65-8.98 cm). These values are significantly higher than those of the untreated group (control), with increased shoot length (17.25 cm), new leaves forming (one leaf) and increased root length (5.56 cm). The contents of chlorophyll a+b in the tested bacterial groups also show higher rates than those of the control group (Table 4).

Chitinase, Protease Activities of Fermented Product by Bacillus megaterium DS9
Bacillus megaterium DS9 demonstrated active antinematodes activity both in vitro and in the greenhouse tests, and also good effect on plant growth promotion. Thus, this strain was chosen for further investigation of active components of its fermented product. To determine the anti-nematode agents produced by this selective strain, we tested the inhibition of supernatant fermented by DS9 against egg hatching and J2 nematodes. Chitinase and protease activity related to the biocontrol of Meloidogyne spp. and anti-nematode activity were also detected.
As shown in Table 5, the culture supernatants of B. megaterium DS9 demonstrated protease (0.69 IU/mL) and chitinase (2.72 IU/mL) activity when mediums were supplemented with casein and chitin, respectively; these culture supernatants also displayed antinematodes J2 activity (52.22-73.33%) and egg hatching inhibition (60-71.11%). The unfermented mediums were also tested activity showing no inhibition against eggs hatching and weak anti-nematode J2 (3.6-8.1%) ( Table 5). After being treated at a high temperature (100 • C) for 60 min, none of the extracted enzymes showed any anti-nematode effect ( Table 6). Enzymatic activity in the culture supernatants disappeared, but the anti-nematode effect still remained in the range of 21.11-30% and 30-37.78% against J2 nematodes and egg hatching, respectively (Table 7).

Isolation, Evaluation of the Active Antinematodes Endophytic Bacteria
Beneficial microbes have been widely used in nematode management and to promote plant growth [10,12,16,[21][22][23]. In this study, beneficial bacteria were isolated then used as environmentally friendly, cost-effective nematode management agents. Unlike previous studies, we focused on isolating bacteria living in the roots of black pepper plants rather than other resources. We chose black pepper fields which suffered heavily from nematodes, and only the roots of healthy black pepper trees surrounded by sick pepper trees (yellow trees affected by root-knot nematodes) were collected for endophytic bacteria isolation. In sick pepper fields cultivated with the same Vinh Linh pepper seedlings, grown under the same conditions, some trees still remain healthy and unaffected by nematodes. This may be due to beneficial microbes in the soil surrounding the roots or living in the roots themselves. Our goal, therefore, was to isolate these microbes. In the current study, we focused on the isolation of endophytic bacteria. Five endophytic strains, DS5, DS8, DS9, DR2, and DR10, demonstrated high anti-nematode activity with 100% mortality. Notably, all of these potentially endophytic bacterial strains belonged to the genus of Bacillus (Figure 2). Various Bacillus species are reported to show anti-nematode activity, such as B. subtilis, B. cereus, B.pumilus, and B. megaterium [20,22,23,33,34]. However, there are few available reports on the anti-nematode activity of B. flexus. Based on recently reviewed literature, this is the first record of endophytic Bacillus species isolated and identified from the Vinh Linh pepper roots in the Central Highlands of Vietnam.
It is interesting to note that all selected endophytic bacteria had a positive effect on black pepper growth (Table 4). Beneficial bacteria may enhance plant growth via several ways, including the inhibition of pathogens and deleterious rhizosphere microorganisms (indirect mechanisms), or supplying growth agents like hormones and nutrients (direct mechanisms) [2,[35][36][37][38]. In this study, Bacillus species displayed potent pathogen inhibition. This bacterial genus has also shown vast beneficial effects, including phosphate solubilization, nitrogen fixation and the production of indole acetic acid, cytokinin, and gibberellin [36]. These may cause the plant growth promotion effect of the selected endophytic bacteria in this study.
To date, vast amounts of genus Bacillus including B. megaterium have been applied for biocontrol and plant growth promotion agents for huge crops [20,22,23,33,34]. However, few studies on B. megaterium applied management of black pepper nematodes as well as their effect on this plant growth promotion.

Primary Determination of Active Components of Fermented Product by Bacillus megaterium DS9
The culture supernatant of B. megaterium DS9 displayed both enzymatic activity (protease and chitinase) and inhibition against J2 nematodes and egg hatching. As such, these tested enzymes may play an important role in anti-nematode activity. To clarify, the crude enzymes were extracted via dialysis, precipitated by ethanol and then tested for anti-nematode activity. As shown in Table 6, the crude enzymes contributed to anti-nematode activity, however this activity disappeared after treatment at high temperature (100 • C) for 60 min. This confirms the enzymes were closely related to anti-nematode activity. These results are consistent with those of previous reports [10,18]. Although the crude extracted enzymes of this bacterium displayed anti-nematode activity (Table 6), inhibition values were lower than those of the original culture supernatants. This may be due to the enzyme extraction process. To confirm this assumption, the culture supernatants were treated at 100 • C for 60 min to denature the enzymes, and then tested for enzyme and anti-nematode activity. The enzymatic activity of the culture supernatants disappeared but the anti-nematode effect remained (Table 7) with low values of 21.11-30% and 30-37.78% against J2 nematodes and egg hatching, respectively. These results prove that the anti-nematode effects were due to enzymes and some other unknown active agents. The culture supernatants showed higher activity than the extracted enzymes due to the other active components. To date, several studies have proven that anti-nematode agents are natural compounds [18][19][20]. Of these, some volatiles are reported as active agents for the control of nematodes [18]. In the current report however, the culture supernatants still maintained anti-nematode activity even after heating. As such, besides enzymes, the major anti-nematode agents must be natural compounds with high thermal stability. Further research is needed to isolate, identify and clarify the role of the major metabolites produced by B. megaterium DS9.

Conclusions
This is the first report on the isolation and selection of endophytic bacteria and utilization for cost-effective control of pepper nematodes in the Central Highlands of Vietnam. Five isolates displayed potent activity with mortality of J2 nematodes value at 100%, and were identified based on 16S rDNA gene sequencing. Of these selective strains, Bacillus megaterium DS9 demonstrated significant antinematode activity in both in vitro and green house tests. This active strain also showed good effect on the promotion of pepper plant growth. The major metabolites produced by Bacillus megaterium DS9 were identified and its enzymatic activities related to the antinematodes effect (chitinase and protease) was also tested. The results suggest that these selected EB strains may be good candidates for the biocontrol of Meloidogyne sp.