Plant growth promoting endophyte Burkholderia contaminans NZ antagonizes phytopathogen Macrophomina phaseolina through melanin synthesis and pyrrolnitrin inhibition

Nazia R Zaman; Umar F Chowdhury; Rifath N Reza; Farhana T Chowdhury; Mrinmoy Sarker; Muhammad M Hossain; Md Ahedul Akbor; Al Amin; Mohammad Riazul Islam; Haseena Khan

doi:10.1371/journal.pone.0257863

. 2021 Sep 30;16(9):e0257863. doi: 10.1371/journal.pone.0257863

Plant growth promoting endophyte Burkholderia contaminans NZ antagonizes phytopathogen Macrophomina phaseolina through melanin synthesis and pyrrolnitrin inhibition

Nazia R Zaman ^1,^#, Umar F Chowdhury ^1,^#, Rifath N Reza ¹, Farhana T Chowdhury ¹, Mrinmoy Sarker ², Muhammad M Hossain ², Md Ahedul Akbor ³, Al Amin ¹, Mohammad Riazul Islam ^1,^*, Haseena Khan ^1,^*

Editor: Abhay K Pandey⁴

PMCID: PMC8483353 PMID: 34591915

Abstract

The endophytic bacterium Burkholderia contaminans NZ was isolated from jute, which is an important fiber-producing plant. This bacterium exhibits significant growth promotion activity in in vivo pot experiments, and like other plant growth-promoting (PGP) bacteria fixes nitrogen, produces indole acetic acid (IAA), siderophore, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. B. contaminans NZ is considered to exert a promising growth inhibitory effect on Macrophomina phaseolina, a phytopathogen responsible for infecting hundreds of crops worldwide. This study aimed to identify the possibility of B. contaminans NZ as a safe biocontrol agent and assess its effectiveness in suppressing phytopathogenic fungi, especially M. phaseolina. Co-culture of M. phaseolina with B. contaminans NZ on both solid and liquid media revealed appreciable growth suppression of M. phaseolina and its chromogenic aberration in liquid culture. Genome mining of B. contaminans NZ using NaPDoS and antiSMASH revealed gene clusters that displayed 100% similarity for cytotoxic and antifungal substances, such as pyrrolnitrin. GC-MS analysis of B. contaminans NZ culture extracts revealed various bioactive compounds, including catechol; 9,10-dihydro-12’-hydroxy-2’-methyl-5’-(phenylmethyl)- ergotaman 3’,6’,18-trione; 2,3-dihydro-3,5- dihydroxy-6-methyl-4H-pyran-4-one; 1-(1,6-Dioxooctadecyl)- pyrrolidine; 9-Octadecenamide; and 2- methoxy- phenol. These compounds reportedly exhibit tyrosinase inhibitory, antifungal, and antibiotic activities. Using a more targeted approach, an RP-HPLC purified fraction was analyzed by LC-MS, confirming the existence of pyrrolnitrin in the B. contaminans NZ extract. Secondary metabolites, such as catechol and ergotaman, have been predicted to inhibit melanin synthesis in M. phaseolina. Thus, B. contaminans NZ appears to inhibit phytopathogens by apparently impairing melanin synthesis and other potential biochemical pathways, exhibiting considerable fungistatic activity.

Introduction

The continued use of expensive chemical fertilizers and fungicides have adversely affected human health and negatively impacted the environment; hence, the safe use of microorganisms that improve soil fertility, enhance plant growth and limit the growth of phytopathogenic fungi has been receiving immense attention from researchers [1]. Endophytes, mainly bacteria and fungi that spend at least a part of their lifespan within plant tissues without negatively affecting their hosts [2], have been intensely studied for years in terms of their diversity, metagenomics, combinatorial biosynthesis, plant growth promotion, biocontrol, bioremediation, etc. [3]. Plant-associated bacteria that aggressively colonize as symbiotic partners in the plant rhizosphere and roots with beneficial effects on plant growth are considered plant growth-promoting rhizobacteria (PGPR) [4]. In contrast, endophytic bacteria colonize the apoplasm or symplasm of the internal tissues of plants [5]. In general, with respect to growth promotion and protection against microbial infection, the beneficial effects of endophytes are considerably greater than those of several rhizobacteria [6].

The endophytes are grouped into three major clusters based on their different mechanisms of action. These contain: (i) biofertilization, which includes siderophore production, atmospheric nitrogen fixation, phosphate solubilization, and exopolysaccharides production; (ii) phyto-stimulation, which includes production of indole acetic acid, gibberellin, cytokinin, and ethylene; and (iii) phyto-biocontrol, which includes competing for iron, nutrients, and space; production of antibiotics, lytic enzymes, volatile compounds; and induction of systemic resistance [7]. Many studies have emphasized the ability of these microorganisms to promote and protect plant growth through additive/synergistic effects [1]; among them, Streptomyces spp. [8], B. subtilis [9], Pseudomonas parafulva, and Pantoea agglomerans [10] are a few examples.

Jute (Corchorus olitorius var. O-4) is an important natural fiber-producing cash crop in Southeast Asia [11]. Throughout its life cycle, jute is confronted by the destructive necrotrophic fungal pathogen Macrophomina phaseolina (Tassi) Goid., which causes charcoal rot disease in more than 500 plant species from approximately 100 families [12].

While screening for jute endophytes with potential inhibitory effects against M. phaseolina, we isolated an endophytic bacterium, Burkholderia contaminans NZ from the seed, which demonstrated promising antifungal activity [13]. This bacterium contained genes that are characteristic of PGPR, such as siderophore and ACC deaminase activities, phytohormone auxin (IAA) production, and nitrogen-fixing abilities, thereby promoting plant growth.

The genus Burkholderia, which belongs to the subphylum of β-proteobacteria, currently consists of 90 validly named species and several uncultivated candidate species [14]. They are ubiquitous organisms with high genetic versatility and adaptability, and are widespread in water, soil, plants, and animals, including humans [15].

However, the Burkholderia genus also comprises certain pathogenic species that threaten plant, animal, and human health [16]. Burkholderia contaminans is a member of the Burkholderia cepacia complex (Bcc) [17] which includes several closely related Burkholderia species. These opportunistic pathogens frequently colonize the lungs of cystic fibrosis (CF) and immune-compromised patients [18]. Furthermore, Bcc is the most frequently isolated clinical pathogen among Burkholderia spp., followed by B. mallei and B. pseudomallei [19]. Therefore, the environmental release of Burkholderia species as a biocontrol agent in agriculture is severely debated due to difficulties in distinguishing plant growth-promoting and pathogenic bacteria [20]. Recent developments in Burkholderia taxonomy and molecular analysis may help in answering questions concerning differences between the pathogenic and ecological properties of Burkholderia species in an attempt to reconsider the possibility of using selected strains for biocontrol [21].

The present study aimed to determine the safety of B. contaminans NZ as a biocontrol agent and its effectiveness against phytopathogens, especially M. phaseolina. The entire genome sequence was analyzed in this study to unravel information regarding the safety of B. contaminans NZ as a PGPR and biocontrol agent based on their siderophores and secondary metabolites producing capacities, and the absence of virulence genes. In vitro data also revealed the attributes of growth promotion. Moreover, Gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) analyses identified several potent antimicrobial compounds and antagonists of melanin synthesis, and the electron transport system produced by the bacterium in the presence of M. phaseolina. These compounds possibly antagonize fungal growth through different modes of action.

Materials and methods

Bacterial, fungal strains and plant materials

The phytopathogenic fungi M. phaseolina was obtained from the Bangladesh Jute Research Institute (BJRI), Dhaka. Nigrospora sphaerica, Xylaria spp., Aspergillus fumigatus, Aspergillus niger, and Penicillium oxalicum were collected from the Molecular Biology Lab, Department of Biochemistry and Molecular Biology, University of Dhaka. Rhizoctonia solani was obtained from the Bangladesh Agricultural University, Mymensingh, Bangladesh. All the fungi were grown and maintained on potato dextrose agar (PDA) and for GC-MS analysis, M. phaseolina was grown in potato dextrose broth (PDB) (HiMedia, India) at 28°C. The antagonistic bacterial strain was isolated from jute seed as an endophytic bacterium as described by Coombs and Franco [22, 23] and identified as B. contaminans by 16S rRNA. Bacterial subculture was maintained on Tryptone Soy Agar (TSA) (HiMedia, India).

Jute seedlings were used to assess plant growth promotion activity of B. contaminans NZ. Fresh seeds of a jute variety (Corchorus olitorius var. O-4) were collected from BJRI. All tests were performed in triplicate if not mentioned otherwise.

Whole genome sequencing of B. contaminans NZ

B. contaminans NZ was cultured overnight in Tryptic Soy Broth (TSB) medium and incubated in an incubator shaker at 37°C. The genomic DNA was extracted using the GenElute™ Bacterial Genomic DNA Kit (Sigma, Germany). A genomic library was constructed and employed for 300-bp paired-end whole-genome sequencing at the Genome Research Institute of North South University, Dhaka, Bangladesh using an IlluminaMiSeq platform (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The generated raw reads (10xcoverage) were assembled using SPAdes version 3.11 [24]. The generated scaffolds were mapped and ordered using ABACAS–a reference-based assembler [25], considering Burkholderia contaminans CH1 as the reference genome [GenBank accession no. AP018357 to AP018360 (four entries)] [26].

Genome sequence analysis and annotation

For structural annotation of the genome Rapid Annotations using Subsystems Technology (RAST) server [27] was used (http://rast.nmpdr.org) along with SEED database for the functional annotation of predicted gene models. SEED also provides subsystem (collection of functionally related protein families) and derived FIGfams (protein families) which represent the core of RAST annotation engine [28]. Subsequently, genome annotation through PIFAR annotation tool was used for the identification of bacterial genetic factors involved in plant host interaction [29]. Moreover, antiSMASH bacterial version 5.0 (https://antismash.secondarymetabolites.org/#!/start) [30] analysis was carried out to identify the biosynthetic gene clusters for secondary metabolites of B. contaminans NZ.

Examining the presence of proteins fostering virulence and pathogenicity

A total of 147 Burkholderia specific proteins reported for pathogenicity and virulence were collected from Virulence Factor Database (VFDB) (http://www.mgc.ac.cn/VFs/main.htm) [31] and investigated for the presence of virulence and pathogenicity related proteins in B. contaminans NZ with the help of BLASTp considering the e-value of at least 1e-10 and sequence identity, not less than 70%.

Data deposition

This Whole Genome Shotgun project has been deposited in DDBJ/ENA/GenBank under the accession QRBC00000000. The version described in this paper is version QRBC01000000.

In vivo growth promotion study of B. contaminans NZ on jute

Inoculation of jute seeds with the endophyte

At first B. contaminans NZ was cultured on TSA plates and then inoculated in TSB media and incubated at 37°C at a shaking speed of 180 rpm for 24–48 h. At the same time, scarification was done for jute seeds with sandpaper and the seeds surface was sterilized using 5% NaOCl. Sterilized seeds were dipped into B. contaminans NZ suspension. From McFarland standard, bacterial cells of 10⁸ CFU/ml were determined by a serial dilution method [32].

In vivo pot experiment in hydroponic culture

Jute seeds inoculated with a bacterial suspension were grown under controlled environmental conditions in a plant growth chamber (Weiss Technik India Private Limited) at 28°C, 70% relative humidity, and 16-h light/8-h dark cycle. The seeds were allowed to germinate for three days and later grown in a hydroponic culture system. 1.25 ml per litre of a modified Yoshida medium (NH₄NO₃ 91.4g, K₂SO₄ 71.4g, NaH₂PO₄.2H₂O 40.3g, CaCl₂.2H₂O 88.6g, and MgSO₄.7H₂O 324.0g per litre) and micronutrients (MnCl₂.4H₂O 1.5g, (NH₄)₆Mo₇O₂₄.4H₂O 0.074g, H₃BO₃ 0.934g, ZnSO₄.7H₂O 0.035 g, CuSO₄ .5H₂O 0.31g, FeCl₃ .6H₂O 7.70 g, citric acid 11.9 g and H₂SO₄50.0g per litre) [33] was added to the seedlings. pH of the solution was adjusted to 5.5 with NaOH. Thirty seeds were sown 1 cm deep in a cork sheet in separate pots. Jute plants were grown for 10 days in hydroponic culture and 5 plants were collected for the measurement of fresh and dry weights, shoot, and root lengths on 4, 7, and 10 days after transfer of the seedlings into the hydroponic solution. The experiment was repeated three times.

Antifungal activity assay

Dual culture assay

In vitro bacteria-fungal dual-culture assays were established by both agar well diffusion and cross streak methods in 9 cm diameter Petri dish systems containing PDA medium [34]. A 5 mm plug taken from the plate of an actively growing fungal colony was inoculated on one side of the Petri dish. Fresh cells of B. contaminans NZ were either streaked in 3 cm length parallel lines on the other side of the fungal plug or 20 μl of the overnight bacterial liquid culture was introduced into a 6 mm diameter well punched aseptically with a pipette tip in an agar plate. Control treatments containing only the fungus were also set up. The plates were incubated at 28°C for 4 to 5 days. The experiment was set up for four replicates. After incubation, the diameters of the fungal colonies were scored and measured.

Antifungal activity of B. contaminans NZ was determined against all the studied phytopathogenic fungi and the toxicity was expressed as a percentage of growth inhibition (PGI) and calculated according to the Zygadlo et al.[35] formula,

P G I (%) = 100 (G C - G T) / G C

where GC represents the average diameter of the fungus grown on PDA (control); GT represents the average diameter of the fungus co-cultivated on the PDA dish with the antagonistic bacterium. A paired t-test was used to check whether the percentage of growth inhibition was significant.

Co-culture of M. phaseolina and B. contaminans NZ

For GC-MS analysis, M. phaseolina was cultured in 20 ml and 100 ml PDB media in two separate conical flasks. B. contaminans NZ was cultured in 80 ml and 100 ml of TSB media in two separate conical flasks.

On the second day of incubation, 20 ml M. phaseolina culture was mixed with 80 ml B. contaminans NZ to form the co-culture and put to incubation again. All the cultures were incubated at 28°C for 5 days for volatile compound extraction and observed for any phenotypic changes in M. phaseolina culture in the presence of B. contaminans NZ.

For HPLC and LC-MS analysis, a separate 1000 ml culture of B. contaminans NZ was cultured under similar conditions described above.

Extraction of organic compounds from culture media

On day 6 of single cultures of fungus, bacterium, and co-cultured media were filtered using Whatman filter paper and centrifuged at 4000 rpm for 7 minutes to remove cell debris. Supernatants were separated into individual conical flasks. 1:1 ethyl acetate (v/v) was added to each flask, shaken, and kept for 4 hours. The organic phases were separated and dried using a rotary evaporator. Finally, the extracts were dissolved in 2 ml of ethyl acetate. A separate extraction (for HPLC and LC-MS analysis) of B. contaminans NZ culture media was also conducted using n-hexane instead of ethyl acetate and shaken for 24 hrs. The extracted fraction was finally dissolved in methanol.

GC-MS analysis of ethyl acetate extract

Ethyl acetate extract of extracellular components was subjected to GC-MS analysis using Shimadzu GCMS-QP2010 Ultra; (Japan) mass detector connected with a capillary column of Rxi-5ms, 30 m long, 0.25 mm i.d.0.25 μm film thickness. 1μL of the sample was injected with splitless mode. Helium was used as the carrier gas with the flow rate was set at 1.0 ml/min. The column oven temperature was programmed from 40°C (1 min hold) at a rate of 10°C/min to 300°C (10 min hold). Injector temperature was maintained at 250°C. Temperatures of the mass interface and ion source were 250°C and 200°C respectively, and detector voltage was 0.4 KV. The analysis was carried out in the EI (electron impact) mode with 70e V of ionization energy. The analysis was performed in full scan mode, ranging from m/z 50 to 400. The compounds were detected after analyzing the mass spectrum of each component using the NIST11 library.

HPLC and LC-MS analysis of methanolic extract

Analytical HPLC Dionex Ultimate 3000 with a C18 column (Nucleodur, 250x4.6 mm, particle size 5μm, pore size 110Å, 100-5C18ec) was used to analyze the bacterial extract dissolved in methanol. After dissolution, the extract was filtered and 200 μL was injected into the column. A multi-step gradient system (solvent system: acetonitrile and water with 0.05% trifluoroacetic acid; flow rate: 1ml/min; temperature: 23°C) was performed with an optimized protocol (0–10 min, 10% acetonitrile; 10–30 min, 10–80% acetonitrile; 30–40 min, 80% acetonitrile; 40–50 min, 80–0% acetonitrile; volume fraction). The wavelength at 225 nm was used to detect compounds eluted from the column. Various fractions were collected containing high peak intensity in the chromatogram and the fractions were dried at low temperature in a lyophilizer (LYOVAC GT 2) and dissolved in methanol to test for antifungal activity and followed by a re-run in HPLC to confirm the reproducibility of the peak. The purified active fraction was then subjected to LC-MS.

For Liquid Chromatography (LC), Agilent 1290 Infinity II instrument was used. C18 column (2.1 mm x 100 mm x 1.8 um) (Zorbax RRHD Eclipse) was used as the stationary phase and 50:50 ratio of water (with 0.1% formic acid) and acetonitrile was used as the mobile phase on isocratic mode. The column temperature and the mobile phase flow rate were kept at 30°C and 0.2 mL/min throughout the program. The sample injection volume was 5 uL. For Mass Spectrometry (MS) analysis, an Agilent 6420 Triple Quad Mass Spectrometer System was used, equipped with an electrospray ionization source operating at positive mode and scanning from m/z 100 to 1000. Nitrogen was used as the nebulizer gas, with pressure and flow set at 45 psi and 11 L/min, respectively. The capillary voltage was maintained at 4000 V, and dry gas temperature at 350°C. Mass Hunter software was used to control and analyze the data. The compound was identified based on its MS fragmentation and analyzed according to literature [36].

Identification of homologous melanin pathway(s) in M. phaseolina

Fungi contain different pathways for melanin biosynthesis; among them two pathways namely 1,8-dihydroxynaphthalene (DHN)-melanin and L-3,4-dihyroxyphenylalanine (DOPA)-melanin pathways, are primarily found in the ascomycota group of fungi [37]. M. phaseolina proteins homologous to this pathway were searched using BLAST. Pathway proteins discovered through literature review and BLAST searches were tested against the M. phaseolina proteome. At least a 30% identity cut-off was taken to consider the homology between the target and query protein [38].

Statistical analysis

The average shoot length, average root length and average height from the three repeated experiment of in vivo pot experiment was taken for statistical analysis. Data obtained were subjected to analysis of variance (ANOVA) test against the control. The results presented as average means, standard deviation (SD), and standard error (SE) were determined by following the standard procedures.

Results

Pot tests for in vivo assay of plant growth promotion

B. contaminans NZ significantly increased the root and shoot lengths, and the average height of jute seedlings (P<0.05) (Fig 1, S1 and S2 Tables). The maximum root length of 11.28 cm, was 116.9% greater than that of the control.

The effect on shoot length was also found to be significant; on average, it was 50.7% longer than that of the control jute plants. Fresh weight of 4-, 7-, and 10-day old jute seedlings increased by 141%, 122%, and 41%, respectively, compared to the controls (P<0.05).

In the presence of B. contaminans NZ, the dry weight of jute plants on an average, increased by 49.8% in all three replicates for each of the 4-, 7-, and 10-day measurements.

Whole genome data analysis

A summary of the whole genome annotation of B. contaminans NZ sequenced using Illumina MiSeq technology is presented in S3 Table. Functional analysis of B. contaminans NZ using the RAST server (Rapid Annotation using Subsystem Technology, http://rast.nmpdr.org), antiSMASH, and PIFAR (plant–bacteria information factor resource) revealed numerous genes and gene clusters (Table 1, S4 Table), which are reported to be exclusively associated with plant growth promotion.

Table 1. List of genes involved in plant growth promotion activity detected from RAST, antiSMASH, PIFAR analysis of the B. contaminans NZ whole genome.

Properties	Name	Biosynthetic genes
Phytohormone production and stress alleviation	Management of ethylene stress	ACC (1-Aminocyclopropane-1-carboxylic acid) deaminase
	Production of IAA (Indole Acetic Acid)	Indole-3-glycerate phosphate synthase
		Indole pyruvate oxidoreductase
		Indole acetamide hydroxylate
		Tryptophan synthase
		Nitrile hydratase
Phosphate solubilization	pyrroloquinoline quinone gene	pqq
Phosphate solubilization	Enolase	eno
Nitrogen fixation	nif gene cluster	nifHDK, nifQ,
Nitrogen fixation	Others	nodT, nir, nor, nolO
Antibiotic biosynthesis
Antibiotic biosynthesis	Pyrrolnitrin	prnA-prnD
Siderophore Biosynthesis	Polychelin	pchR
	Ferric siderophore transport	pchD-pchA
	ABC-type siderophore export system	feoB
	Siderophore pyoverdine	pvd

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As observed in other PGPR Burkholderia strains [39], the nifHDK operon required for nitrogen fixation was detected in B. contaminans NZ, along with the 1-aminocyclopropane-1-carboxylate (ACC) deaminase coding sequence, gene coding for iron(III) ABC transporter substrate-binding protein, phytoene synthase gene, the coding sequence for phosphotransferase system (PTS), and major facilitator superfamily (MFS) transporter genes. In silico analysis using RAST also revealed the presence of genes for indoleacetamide hydrolase involved in IAA production, which is a plant hormone associated with plant growth [40], and the eno gene, which assists in phosphate solubilization. Genome analysis also indicated the presence of pyrroloquinoline quinone synthase and glucose dehydrogenase, which are implicated in gluconic acid and 2-ketogluconic acid production, and required for mineral phosphate solubilization [41]. The annotated genome of B. contaminans NZ also contains 30 siderophore-related genes associated with ferric siderophore transport, ABC-type siderophore export system, arthrobactin, siderophore pyoverdine, and an intact siderophore pyochelin biosynthesis pch gene cluster. From the PIFAR data, in B. contaminans NZ, the biosynthesis of the major siderophore pyochelin is apparently associated with five genes (PMID: 22261733) distributed over five different regions in the bacterial genome.

While examining the core biosynthesis genes, genetic loci related to antibiotic production were also found in B. contaminans NZ, as presented in Table 1. Moreover, genes for pyrrolnitrin, an antifungal secondary metabolite produced by certain Bcc and several other gram-negative bacteria, were also identified. This metabolite inhibits the growth of a wide range of fungi and pathogenic bacteria [42, 43].

Most virulence genes absent in B. contaminans NZ

Various virulence factors reported in pathogenic bacteria and other plant endophytic strains of Burkholderia (B. contaminans CH1, B. contaminans MS14) were screened for in the B. contaminans NZ genome, which failed in identifying major virulence-related genes, such as the biosynthesis genes for cable pili, toxoflavin, cepacian, and O-antigen (O-Ag) biosynthetic cluster (Table 2). This differs from pathogenic strains that contain many virulence genes. However, few virulent genes in B. contaminans NZ are present as incomplete gene clusters.

Table 2. List of major virulence related genes present or absent in B. contaminans NZ.

Feature name	Relevant gene/gene cluster	Status
Actin based intracellular motility	BimA	Absent
Adherence	BoaA	Absent
	BoaB	Absent
	Type VI pilin system	Incomplete cluster (3 out of 11 genes)
Anti-phagocytosis (O antigens)	Capsule I gene cluster	Absent
Secretion system	Bsa T3SS cluster	Absent
Secretion system	T6SS-1 cluster	Absent
Signaling	Cdp	Present
Signaling	Quorum sensing	Incomplete cluster (2 out of 8 genes)
Cepacian	bceA-bceK, bceN- bceT	Absent
Cable pilin gene	cblA	Absent
Toxoflavin	toxR, toxA-toxE	Absent
2-heptyl-3-hydroxy-4(1H)-quinolone	pqsA-pqsE	Absent
Hydrogencyanide	hcnA-hcnC	Absent
Cu2+ and Zn2+ containing periplasmic SOD	apaH-reG	Present
Zinc metalloprotease	Zmp	Present

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Antifungal activity assay

In dual culture, B. contaminans NZ substantially inhibited the growth of six plant fungal pathogens, namely Nigrospora sphaerica, Xylaria spp., Aspergillus fumigatus, Aspergillus niger, Penicillium oxalicum, and Rhizoctonia solani (Fig 2).

Fig 2 — *(a) Nigrospora sphaerica* (b) *Xylaria* spp.(c) *Aspergillus fumigatus* (d) *Aspergillus niger* (e) *Penicillium oxalicum*, and(f) *Rhizoctonia solani*.

In dual-culture assay, B. contaminans NZ demonstrated significant growth inhibitory activity, which ranged from 44% to 58% than that of the control (P<0.05), when co-cultured with all the tested fungi. The growth inhibition rates observed for each fungal species are shown in Table 3.

Table 3. Growth inhibition induced by B. contaminans NZ on different plant pathogenic fungi.

Fungal species	Growth inhibition (%) (Mean ± SD)
Xylaria spp.	57.71 ± 1.88
Aspergillus fumigatus	45.94 ± 1.45
Aspergillus niger	54.38 ± 4.10
Penicillium oxalicum	55.64 ± 2.96
M. phaseolina	49.04 ± 3.41
Nigrospora sphaerica	52.99± 3.67
Rhizoctonia solani	44.31 ± 4.36

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Growth suppression and chromogenic aberration in B. contaminans NZ challenged M. phaseolina

M. phaseolina inoculated in liquid culture was initially pale in color, which assumes a characteristic black color within two to three days that perpetuates until day 5. However, a M. phaseolina culture challenged by B. contaminans NZ deviates from this usual behavior and retains the initial pale color up to day 5. Furthermore, M. phaseolina growth when subjected to co-culture with the bacterium remained practically static from day 2. Fig 3 shows the phenotypic changes in fungal growth from days 2 to 5.

Fig 3 — (A) M. *phaseolina*, B. *contaminans* NZ, and their co-cultures on day 5. (B) M. *phaseolina* without B. *contaminans* NZ retains the black color (left) compared to its B. *contaminans* NZ challenged counterpart (right). Inhibition of M. *phaseolina* growth and its pigmentation in the presence of B. *contaminans* NZ is evident compared to the culture containing only M. *phaseolina*.

Chemical analysis of bio-active compounds using GC-MS

GC coupled with MS was used to identify the organic compounds produced by the fungus, bacteria, and their co-culture, and to identify the chemical nature of the bioactive compounds responsible for inhibiting fungal growth. Three sets of secondary metabolites obtained from (i) B. contaminans NZ, (ii) M. phaseolina, and (iii) B. contaminans NZ -M. phaseolina dual culture aided in identifying more than 72 compounds. Among them, 9,10-dihydro-12’-hydroxy-2’-methyl-5’-(phenylmethyl) ergotaman 3’,6’,18-trione; 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one; 1-(1,6-dioxooctadecyl)- pyrrolidine; 9-octadecenamide; 3-trifluoroacetoxypentadecane;2-hexyldecanol; and catechol, reportedly exhibit various biological activities, including tyrosinase inhibition, antifungal, and antimicrobial effects (Table 4). For example, catechol acts as a suicide inhibitor of enzyme tyrosinase [44], which forms melanin in fungi. Furthermore, 9, 10-dihydro-12’-hydroxy-2’-methyl-5’-(phenylmethyl) ergotaman 3’, 6’, 18-trione acts as an antimicrobial and anti-inflammatory agent [45]. A partial list of compounds and their corresponding functions are provided in Table 4, and peaks from GC-MS analyses of M. phaseolina (M), B. contaminans NZ (B), and M. phaseolina and B. contaminans NZ co-culture (C) are shown in Fig 4.

Table 4. List of potential compounds with their biological activities and retention times found in GC-MS analysis of B. contaminans NZ (B), M. phaseolina (M), and their co-culture (C).

Sl. No.	Compound Name	Culture Name	Retention Time (min)	Biological Activity	Reference(s)
1	2,5-Dimethyl pyrazine	B, C	6.28	Fungistatic activity	[46]
2	2-Methoxy- phenol	C	9.30	Antioxidant, Cytotoxic	[47]
3	5-Methyl-furfural	M, C	7.1	Antimicrobial, Antioxidant	[48]
4	2-Undecanethiol, 2-methyl	M, C, B	9.01/12.6	Antimicrobial activity	[49]
5	2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one	B, C	10.4	Antifungal, Oxidative stress inducer	[50, 51]
6	Catechol	C	11.4	Antifungal activity, Suicide substrate inhibitors of tyrosinase	[44, 52, 53]
7	1,4-Benzenediol / Hydroquinone	B	11.42	Cytotoxic activity, Protein kinase inhibition	[54]
8	2-(1,1-Dimethylethyl)-4,6-dinitro-phenol	B	12.8	Herbicide	[55]
9	Decahydro-1,4-dimethoxy naphthalene	B	14.6	Antimicrobial activity	[56]
10	2-Hexyldecanol	C	16.06	Suppresses melanin synthesis	[57]
11	3-trifluoro acetoxypentadecane	C	16.6	Antimicrobial activity	[58]
12	Methoxyacetic acid, 3-tridecyl ester	M, B	16.6/17.8	Cytotoxicity	[59]
13	Pyrrolo[1,2-a]pyrazine-1,4-dione	M, B	19.6/19.9	Antioxidant agent, Antifungal activity, Antibiotic activity	[60–62]
14	3,6-Diisopropylpiperazin-2,5-dione	B	20.1	Antimalarial activity	[63]
15	1-(1,6-Dioxooctadecyl)- pyrrolidine	B, C	20.23/21.2	Antioxidant activity	[64]
16	n-Hexadecanoic acid	B, M, C	21.04	Antibacterial, Antifungal, Cytotoxicity,	[65]
17	Hexadecamethylheptasiloxane	B, C	23.8	Antifungal	[66]
18	9-Octadecenamide	B, C	24.8	Antimicrobial, Anti-inflammatory	[58]
19	9,10-Dihydro-12’-hydroxy-2’-methyl-5’-(phenylmethyl)-, (5’ alpha,10 alpha) - ergotaman-`3’,6’,18-trione	B, C	24.9/25.1	Antimicrobial, Anti-inflammatory, Alpha-amaylase inhibitory activity	[45]
20	Di-n-octyl phthalate	B, M	25.9/ 26.35	Antifungal and antioxidant	[67, 68]

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Fig 4 — (A) M. *phaseolina*, (B) M. *phaseolina* and B. *contaminans* NZ co-culture, and (C) B. *contaminans* NZ. The number on different peaks corresponds to the serial number of the compounds in **Table 4**.

Identification of pyrrolnitrin in methanolic extract of B. contaminans NZ culture

The chromatogram of RP-HPLC analysis of the methanolic crude extract showed various peaks (Fig 5A). RP-HPLC fractions were collected and their activity tested against M. phaseolina, wherein the fraction obtained at a retention time of 33.2 min exhibited antifungal activity (Fig 5B). The compound with a molecular ion peak of 257.1 (m/z) was identified as pyrrolnitrin via LC-MS analysis, after comparing with standard pyrrolnitrin (Fig 5C).

Identification of putative melanin pathways in M. phaseolina through homology-based search

After an extensive literature review of different melanin pathways active in fungi, polyketide synthase, tetrahydroxy naphthalene reductase, trihydroxy naphthalene reductase, scytalone dehydratase, and tyrosinase proteins were selected, and their corresponding homologs in M. phaseolina were identified through a BLAST search. The details are presented in Table 5. In addition, putative pathways proposed in S1 Fig presents the order in which the reactions occur during melanin synthesis in M. phaseolina.

Table 5. Homologous proteins of melanin pathways found in M. phaseolina.

Query Accession	Query Name	Subject Accession	Subject Name	Percent Identity
K2S0W9	Betaketoacyl synthase	D7RJP3	Polyketide synthase	31.25
K2RKI0	Short-chain dehydrogenase/ reductase SDR	Q12634	Tetrahydroxy naphthalene reductase	75
K2RKI0	Short-chain dehydrogenase/ reductase SDR	W3XC32	Trihydroxy	75.5
			Naphthalene
			reductase
K2R7Y4	Scytalone dehydratase	W3XEE6	Scytalone	82.7
K2R7Y4	Scytalone dehydratase	W3XEE6	dehydratase	82.7
K2R777	Tyrosinase	U7PL38	Tyrosinase	31.132

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Discussion

Since chemical control and most cultural practices are not effective representative tools that limit the pathogenic fungal growth and their distribution [69], the use of biocontrol agents has become a promising alternative in agriculture. Biocontrol using plant growth-promoting rhizobacteria (PGPR) is a potentially attractive and efficient disease management approach, as it promotes plant growth, enhances tolerance to abiotic stress [70], suppresses pathogens locally, and induces systemic resistance (ISR) against a broad range of crop diseases [71, 72].

Several studies conducted on endophytes have emphasized their ability in promoting plant growth and their additive/synergistic effects on plant growth and protection. In general, such growth-promoting rhizospheric bacteria belong to the following species: Alcaligenes, Arthrobacteria, Azospirillum, Azotobacter, Bacillus, Burkholderia, Enterobacter, Klebsiella, and Pseudomonas [8, 73]. The endophytic strain B. contaminans NZ isolated from jute has also been characterized in vivo for its potential plant growth-promoting (PGP) traits, which significantly increased both yield and biomass when jute seedlings were inoculated with the bacterium, and notably reduced the development of disease symptoms caused by M. phaseolina.

As B. contaminans NZ exhibited growth promotion and apparent protection against the phytopathogen M. phaseolina, its whole genome was sequenced to determine the characteristic genetic features. Genome annotation of B. contaminans NZ assisted in identifying genes related to phytohormone production, stress alleviation, phosphate solubilization, nitrogen fixation, antibiotic biosynthesis, and siderophore biosynthesis. In vitro experiments of the endophyte also displayed significant production of the above-listed compounds (S5 Table, S2 Fig).

Certain Burkholderia species demonstrate clinical associations and plant-pathogenic traits [74, 75]; therefore, genes related to virulence and pathogenicity were also investigated in the B. contaminans NZ genome. Several bacterial species from the B. cepacia complex (Bcc) are opportunistic pathogens [76]. B. cenocepacia strains expressing both cable (Cbl) pili and 22-KDa adhesin proteins have been reported to bind strongly to cytokeratin 13 (CK13), which efficiently infects the host cell by invading the squamous epithelium [77]. However, the cblA (giant cable pili) gene was absent in the symbiotic and legume-nodulating species [78]. B. contaminans NZ also lacks the cable pili biosynthesis gene cluster, indicating their inability to attach to the host cell, which is required to initiate infection. Furthermore, VgrG-5, which is a Burkholderia type VI secretion system 5-associated protein, required for complete mammalian virulence [79], is also absent in B. contaminans NZ genome. Virulence-associated protein sequences (for chemotaxis, attachment, type 3 and type 6 secretion systems; T3SS and T6SS) and typically described hallmark features representing the true and opportunistic pathogenic Burkholderia strains are also absent in B. contaminans NZ. Cystic fibrosis (CF)-related O-antigen of lipopolysaccharides associated with transmitting infections in CF patients [80] and zinc metalloproteases that may be involved in the overall virulence of several Bcc strains [81] are also absent. Among the virulence-associated proteins of Burkholderia, only the sodC gene encoding a Cu²⁺ and Zn²⁺ containing periplasmic superoxide dismutase that contributes in intracellular survival, which indicates self-protection ability in CF patients, is present in the genome [82]. Burkholderia species commonly produce plant-toxic secondary metabolites, polysaccharides, and other toxins such as rice grain rot and wilt causal agent, toxoflavin; exo-polysaccharide toxin, cepacian; hydrogen cyanide (HCN); and 2-heptyl-3-hydroxy-4(1H)-quinolone [80]. These pathogenic genes were absent in this jute endophytic bacterium.

The B. contaminans NZ genome also contained antibiotic-biosynthetic genes. Thus, to gather further evidence, M. phaseolina and several other phytopathogenic fungi, namely, Xylaria spp., Aspergillus fumigatus, Aspergillus niger, Penicillium oxalicum, Nigrospora sphaerica, and Rhizoctonia solani were co-cultured with B. contaminans NZ, where all fungi tested were suppressed with varying degrees of inhibition (Fig 2, Table 3), confirming its antifungal activity.

Growth inhibition of M. phaseolina by B. contaminans NZ caused marked morphological changes and chromogenic aberration in the phytofungus [13]. Such morphological changes in the cell membrane have also been reported for B. cepacia-mediated inhibition of F. solani and C. dematium [83]. Burkholderia CF66I noticeably alters R. solani hyphae with multiple branches and swelling [84]. In previous reports, co-culturing M. phaseolina and B. contaminans NZ in solid media caused the fungus to lose its characteristic black color, restricted its growth, and attenuated its infectivity [13]. The present study also observed a similar deviation in pigmentation and growth repression in liquid co-culture (Fig 3).

Various reports have demonstrated that secondary metabolites produced by certain bacteria can influence fungal growth [85, 86]. Therefore, secondary metabolites were analyzed to explain chromogenic aberration and growth suppression of M. phaseolina when co-cultured with B. contaminans NZ. GC-MS analysis of ethyl acetate extracts of culture supernatant revealed over 72 compounds, among which 20 compounds are biologically important; 2,5-dimethyl pyrazine, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, hexadecamethylheptasiloxane, 9-octadecenamide, and 9,10-dihydro-12’- hydroxy-2’-methyl-5’- (phenylmethyl)-, (5’ alpha,10 alpha)- ergotaman-3’,6’,18- trione compounds were discovered in both B. contaminans NZ and its co-cultures, but were absent in the M. phaseolina extract. 2,5-dimethylpyrazine, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, and hexadecamethylheptasiloxane exhibits antifungal activities [46, 50, 51, 87]. In addition, 9-octadecenamide and 9,10-dihydro-12’- hydroxy-2’-methyl-5’- (phenylmethyl)-, (5’ alpha,10 alpha)- ergotaman-3’,6’,18- trione reportedly exhibits antimicrobial properties [58, 88]. The absence of these compounds in M. phaseolina extract implies that they are most likely produced only by B. contaminans NZ, thereby inhibiting the pathogen in the liquid media. 2-hexyldecanol, which suppresses melanin biosynthesis [57], and catechol, which demonstrates antifungal properties [52], are exclusively produced in the co-culture. Catechol, is also known as a suicide substrate for enzyme tyrosinase [53], which is involved in the DOPA-melanin biosynthesis pathway [37]. These compounds apparently produced only by B. contaminans NZ during co-culture condition are expected to play a role in the chromogenic aberration and growth suppression of M. phaseolina. n-Hexadecanoic acid possesses antifungal, antibacterial, and cytotoxic activities [65] and 2-undecanethiol, 2-methyl possesses antimicrobial activity [49]. Both compounds are ubiquitously present in B. contaminans NZ, M. phaseolina, and their co-cultures. Both organisms appear to be armed with antagonistic compounds, ready to inhibit each other in an attempt to avail the available resources.

Burkholderia spp. reportedly produces potent antifungal compound pyrrolnitrin [89]. This compound exhibits inhibitory effects by obstructing the synthesis of vital biomolecules (DNA, RNA, and protein), uncoupling oxidative phosphorylation, impeding mitotic division, and inhibiting several biological mechanisms. The genome of B. contaminans NZ also contained a gene cluster for pyrrolnitrin synthesis (Table 2). Although the GC-MS data revealed the presence of several compounds that implied possible modes for M. phaseolina growth suppression, pyrrolnitrin in the secondary metabolite profile was absent. Therefore, the extraction process was altered by substituting the extraction solvent with n-hexane instead of ethyl acetate to identify pyrrolnitrin, which is in agreement with earlier reports that employed a similar method [43]. Thereafter rest of the method remained similar except that the extract was dissolved in methanol instead of ethyl acetate. RP-HPLC was performed to purify the active compound(s) to detect the appreciable suppression of fungal growth by the crude extract. Only one fraction exhibited considerable inhibitory activity against M. phaseolina, and its mass was identified using LC-MS analysis (257.1 Da), which was identical to the standard pyrrolnitrin (molecular weight: 257.07 g/mol).

A previous iTRAQ proteomic analysis of M. phaseolina challenged with Burkholderia showed the downregulation of beta-ketoacyl synthase, scytalone dehydratase, tyrosinase, enzymes of the DHN-melanin, and DOPA-melanin pathways [13]. Catechol present in the co-culture of B. contaminans NZ and M. phaseolina can explain the downregulation of tyrosinase. Kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] is a prominent tyrosinase inhibitor [37], and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, identified by GC-MS analysis, is an oxidized form of kojic acid and is expected to act identical to its reduced counterpart. This derivatization could have occurred during sample processing for GC-MS analysis.

Further investigation of the M. phaseolina enzymes involved in melanin synthesis, similar to those reported earlier in other fungi, led to the identification of SDR–short chain dehydrogenase/reductase, which is similar to both trihydroxy naphthalene reductase [90] and tetrahydroxy naphthalene reductase [91], which are enzymes of the DHN-melanin pathway [92]. Tyrosinase [92, 93] performs multiple catalysis in the DOPA-melanin pathway; it converts phenylalanine to tyrosine, tyrosine to DOPA, and DOPA to DOPA quinone, which is eventually converted to melanin further downstream. However, both DOPA-melanin and DHN-melanin pathways are yet to be fully elucidated. This reduces the scope of finding other homologous pathway proteins in M. phaseolina. The percent identity of beta-ketoacyl synthase and tyrosinase was lower (~31%) than that of tetrahydroxy naphthalene reductase, trihydroxy naphthalene reductase, and scytalone dehydratase (~75% or more). This was possibly due to only few reports that describe the two genes with precise genomic features. Based on this evidence, a tentative melanin pathway was proposed for M. phaseolina in this study (S1 Fig).

Conclusion

Whole genome analysis and secondary metabolite characterization have acknowledged B. contaminans NZ as a good biocontrol agent, in addition to its role in plant growth promotion. It suppresses the growth of multiple fungi, especially the phytopathogen M. phaseolina, by using catechol, pyrrolnitrin, and other antimicrobial agents. Compounds identified in the extracts of B. contaminans NZ or the co-culture of B. contaminans NZ and M. phaseolina contain compounds that inhibit melanin biosynthesis, which possibly contributes to the observed chromogenic aberration and growth suppression of the fungi. B. contaminans NZ can be established as a bio-control agent by conducting toxicity tests to ensure its safety, followed by field experiments to determine its efficacy under different environmental conditions. Hence, further studies are needed to optimize the formulation and application methods of B. contaminans NZ to fully maximize its potential as an effective agent in controlling M. phaseolina.

Supporting information

S1 Fig. Proposed melanin pathways in M. phaseolina based on homology search.

(TIF)

Click here for additional data file.^{(475.1KB, tif)}

S2 Fig

Qualitative assay for (a) siderophore, (b) ACC deaminase, and (c) nitrogen of B. contaminans NZ.

(TIF)

Click here for additional data file.^{(112.9KB, tif)}

S1 Table. Average root length, shoot length, and plant height of bacteria treated jute seedlings vs. untreated control in a pot experiment at 4, 7, and 10 days.

The data of three replicates per experiment are presented as means and standard deviations.

(DOCX)

Click here for additional data file.^{(17KB, docx)}

S2 Table. Fresh weight and dry weight of the bacteria treated jute seedlings vs. untreated control in a pot experiment at 4, 7, and 10 days.

The data of three replicates per experiment are presented as mean(s) and standard deviation(s).

(DOCX)

Click here for additional data file.^{(15.4KB, docx)}

S3 Table. Genomic features of Burkholderia contaminans NZ.

(DOCX)

Click here for additional data file.^{(14.1KB, docx)}

S4 Table. Location of genes involved in plant growth promotion activity detected from RAST, antiSMASH, and PIFAR analysis of the whole genome of B. contaminans NZ.

(DOCX)

Click here for additional data file.^{(19.1KB, docx)}

S5 Table. In vitro plant growth promotion attributes of B. contaminans NZ.

(DOCX)

Click here for additional data file.^{(17.3KB, docx)}

Acknowledgments

The authors cordially thank NSU Genome Research Institute, North South University, Bangladesh for the whole genome sequencing and Dr. Abdul Baten, AgResearch Ltd. Christchurch, NZ for helping with the whole genome analysis and Enayet Hossain for collecting the GC-MS data. We also acknowledge the Bangladesh Council of Scientific and Industrial Research (BCSIR) for providing support to carry out the GC-MS.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This project is funded by Higher Education Quality Enhancement Project (HEQEP) (Grant number: CP-3250), a World Bank financed development project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0257863.r001

Decision Letter 0

Abhay K Pandey

30 Jun 2021

PONE-D-21-18655

Plant growth promoting endophyte Burkholderia contaminans NZ antagonizes phytopathogen Macrophomina phaseolina through multiple modes of action

PLOS ONE

Dear Dr. Islam,

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Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: On the basis of what the authors reported in the manuscript, the present study was aimed to "Plant growth promoting endophyte Burkholderia contaminants NZ antagonizes phytopathogen Macrophomina phaseolina through multiple modes of action".

To gain these aims, the authors developed series of experiments devoted to re-culturing, identify pathogen and growth promoting bacterial isolates using GC-MS and whole genome sequencing and the information in results is clear and concise but need to be checked again as there are few grammatical error and used a few long sentences that should be summarized.

Materials and methods should be more attractive if the sequencing and analysis be placed before the in-vivo and in-vitro studies; over all the manuscript is written well and have valuable information for the readers. The whole manuscript should be proofread for English language editing and grammatical errors.

Discussion section of the manuscript is should be improved by using a few latest references.

Reviewer #2: 1. Objective of the study should be more clearly defined.

2. Replicates used in the experiments are very few, so my question is why the authors did not repeat the experiments. If they did, then it requires proper elaboration.

and How will authors justify the findings and their reproducibility?

3. Discussion is very very long, it should be précised.

4. Conclusion section is lacking impressiveness in its expression. I will suggest authors to put an extra effort in improving it.

5. There is also an suggestion about the title, I think it should be more specific as it is very generalized in its current form.

**********

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Reviewer #1: Yes: Raees Ahmed

Reviewer #2: No

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PLoS One. 2021 Sep 30;16(9):e0257863. doi: 10.1371/journal.pone.0257863.r002

Author response to Decision Letter 0

29 Jul 2021

Responses to the Editor and Reviewers’ comments

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

The format has been changed according to PLOS ONE’s style.

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

We would like to address the funding information as stated below:

We will update your Data Availability statement to reflect the information you provide in your cover letter.

There are no ethical or legal restrictions to sharing our data publicly. Data Availability statement has been corrected to ‘The authors have no objection to make the data set underlying the results described in our manuscript to be fully available.’

In the revised cover letter the accession no of the bacterial whole genome sequence (This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession QRBC00000000) and data availability statement have been included.

An ORCID iD was generated and validated in Editorial Manager

The results have been included in the Supporting Information (S2 Fig and S5 Table)

6. We note that Figures 1, 2, and 3 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

There is no chance of copyright issue for Figures 1, 2 and 3. They have been produced in this study and have not been used in any other article or report.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

5. Review Comments to the Author

Reviewer #1:

On the basis of what the authors reported in the manuscript, the present study was aimed to "Plant growth promoting endophyte Burkholderia contaminants NZ antagonizes phytopathogen Macrophomina phaseolina through multiple modes of action".

Discussion section of the manuscript is should be improved by using a few latest references.

Changes has been made according to the suggestions.

Reviewer #2:

1. Objective of the study should be more clearly defined.

The objective has been clearly defined.

2. Replicates used in the experiments are very few, so my question is why the authors did not repeat the experiments. If they did, then it requires proper elaboration and How will authors justify the findings and their reproducibility?

All the experiments were done with three replications as stated in the Materials and Method section (lines 119 and 172) and all tests were performed in triplicate if not mentioned otherwise.

3. Discussion is very long, it should be précised.

Discussion has been modified accordingly.

4. Conclusion section is lacking impressiveness in its expression. I will suggest authors to put an extra effort in improving it.

This has been modified accordingly.

5. There is also an suggestion about the title, I think it should be more specific as it is very generalized in its current form.

The title has been edited as suggested.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Raees Ahmed

Reviewer #2: No

Reviewer Recommendation Term:

Major Revision

1. Is the manuscript technically sound, and do the data support the conclusions?

Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Yes

6. Review Comments to the Author

The data itself is good, but the manuscript need to be improved in terms of writing. There are so many small mistakes that can easily be fixed by just reading it through a few more times. Here I listed a portion of the problems, but I recommend the authors to go over carefully for each sentence and craft it again. Also, I didn't like the red background for some figures. Although I won't say this is mandatory, I think red is probably not the best background color for those figures.

Overall, this paper presents solid data sets and I recommend it to be published after major revisions.

Abstract

Line 40- Secondary metabolites, catechols and ergotaman (that has been found through what?)

They were found through GC-MS analysis as mentioned in line 35.

Introduction

Line 58- be specific of what 'They' indicates (ex: the beneficial effects of endophytes)

Revised accordingly

Line 61-63

biocontrol activity including competition for iron, nutrient and space, production of antibiotics, lytic enzymes and volatile compounds, and induction of systemic resistance.

Corrected accordingly

Line 63-65

Many studies have emphasized the ability of these microorganisms for possible roles for promoting plant growth and protection through additive/synergistic effects.

Corrected accordingly

Line 66

B. subtilis [9], Pseudomonas parafulva, and Pantoea agglomerans are a few examples.

Not so sure about "recent developments". What developments?

Corrected accordingly

Line 68

Full scientific name for Jute

Throughout its life cycle,

Corrected accordingly

Line 81

human environment? Like inside of human body? If so,

"a widespread presence in water, soil, plants, and animals including human"

Corrected accordingly

Line 84 & 86

Space between (Bcc) and [17] / [18] and .

Corrected accordingly

Line 90

Don't start a line with "but"

Corrected accordingly

Line 94-96

I think this line can be written shorter and clearer.

Changed accordingly

Overall, introduction is good, but can be better by reorganizing and rewriting sentences.

Line 105

Delete "used in this study ... collection of"

Corrected accordingly

Materials and methods

Line 108-109 and throughout manuscript

Use full scientific names including authroties if possible.

These should be okay.

Line 117

Fix 'NZ' (italic)

Corrected accordingly

Line 123

At first,

TSB media? Full names should be mentioned when first introduced. Instead of that, full name was introduced in line 199

Corrected accordingly

Line 124

At the same time,

Corrected accordingly

Line 134

and MgSO4.7H2O

Corrected accordingly

Line 137

Thirty instead of 30

Corrected accordingly

Line 152

(10x coverage)

Corrected accordingly

Line 158

server [29]

Corrected accordingly

Line 198 and throughout manuscript

Authors should be consistant for number+unit (ul, degree, etc) combination. For example, I can see inconsistancies such as 20ml (no space) vs 20 ml (space between number+unit). Just go with one.

Corrected accordingly

Line 205-206

For HPLC and LC-MS analysis, a seperate 1000ml of B. contaminans NZ was cultured under similar conditions described above.

Corrected accordingly

Line 208

Delete 'together' since co-culture already contains the meaning.

Corrected accordingly

Line 209

filtered thorough 'what'?

Corrected accordingly

Line 213-215

Recommend to rephrase it. Hard to understand.

Revised accordingly

Line 220

One µL is awkward. What about one microliter?

Corrected accordingly

Line 221

Helium was used as the carrier gas with the flow rate set at 1.0 mL/min

Also, be comsistant with ml or mL (chose one)

Corrected accordingly

Line 224

respectively, and

Corrected accordingly

Line 235

temperature:

Corrected accordingly

Line 251-252

The capillary voltage was maintained at 4000 V, and dry gas temperature was set at 350°C.

Corrected accordingly

Line 256

biosynthesis; among those,

Corrected accordingly

Results

Table 1.

Adding chromosomal locations for the genes would be beneficial.

Has been added in S4 Table

Line 334

and (f) Rhizoctonia solani

Corrected accordingly

Table 3.

Xylaria sp.

Nigrospora sphaerica 52.99 ± 3.67

Corrected accordingly

Line 403-404

, and their 404 corresponding homologues in M. phaseolina were identified through a BLAST search

Corrected accordingly

Discussion

Line 416

Delete ,

Corrected accordingly

Line 440

Delete But and replace with 'however' or similar word.

Corrected accordingly

Line 458

The Burkholderia genome

Corrected accordingly

Line 459

M. phaseolina

Corrected accordingly

Line 461

and Rhizoctonia solani

Corrected accordingly

Line 481

Space issue

Corrected accordingly

Line 530

, and scytalone dehydratase

Corrected accordingly

Line 547

downregulation

Corrected accordingly

Attachment

Submitted filename: Responses to the reviewers comment.docx

Click here for additional data file.^{(34.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0257863.r003

Decision Letter 1

Abhay K Pandey

16 Aug 2021

PONE-D-21-18655R1

Plant growth promoting endophyte Burkholderia contaminans NZ antagonizes phytopathogen Macrophomina phaseolina through melanin synthesis and pyrrolnitrin inhibition

PLOS ONE

Dear Dr. Islam,

Please submit your revised manuscript by Sep 30 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Abhay K. Pandey

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

This manuscript is an improvement over the previously submitted version. Although all reviewers recommended publication of the MS, however, The MS still needs improvement in terms of clarity and quality. I recommended in my last review that the authors find someone fluent in scientific writing in English to correct the many grammar, terminology, and sentence structure errors in the manuscript. While, some effort was made to correct these errors, the current manuscript is still filled with errors that make the article very difficult to read. I have noted a few of these errors on the manuscript, but there are many errors per page that need to be addressed. The level of writing is not up to the standard required for publication in PLoS series journals. At this point, the authors should consider employing a professional editor experienced in scientific writing.

In abstract, background of research and aim of the study are missing, please revise abstract to make it more focused.

What is NZ in abstract, this is a wrong way of presentation please revise, do you mean Burkholderia contaminans, if it is, then it is not properly cited.

Page 2 lines 34 compounds or molecules not substances, and overall the MS need thorough English revision failing which MS will be not accepted. Overall, abstract is very poorly written, please revise it to make more focused.

Page 3 line 65 line 85 species not italic correct in whole MS

Line 77 space

Line 92 to 101 it should described objective of your research and why it needed and then what are you presenting in this paper.

Did you check pathogenicity of Macrophomina phaseolina before conducting experiments?

Line 120 it was not grown in shaker, you incubated in incubator shaker, how long old culture you used for DNa isolation

Line 157 spore density was adjusted ??? revise sentence

Line 172, the experiment was repeated three times, if it is the case, how you handled the data from repeated experiments it is not described in the statistical section. Also this section does not show you used original data or transformed data for analysis.

Petri dish, P should be capital

Line 193 35 and 36 why two reference, anyway this paper contains more references, more than we require for a research paper, please reduce the number of references upto 50.

Remove statistical part from each section and put it in the last of material and method in the single section describing the details how u analyzed the data as I mentioned.

Line 259 ascomycota group of fungi

Line 272 (P<0.05)

Table1 I think name of genes should be italic, follow in all tables

Mention CD, MSS and F values in table 3.

Coulumn one Fungal Species

Line 342 full name Macrophomina phaseolina should start follow in whole in whole MS same trend

Table 4 can be given as supplementary table

Line 441 strains

Conclusion should be short and easily understandable

Some of the references not follow the journal trends.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: I have received and checked critically the revisions from the author on the the manuscript with title "Plant growth promoting endophyte Burkholderia contaminans NZ antagonizes phytopathogen Macrophomina phaseolina through melanin synthesis and pyrrolnitrin inhibition" and found satisfactory.

On the light of this my recommendation is to accept the manuscript keeping in view the decision of other experts.

Reviewer #2: (No Response)

Reviewer #3: Authors corrected listed errors/problems, and this MS looks a lot better now.

Just a few recommendations:

Based on track change file

Line 81: There is a space between [14] & .

Line 125-126: What rpm?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Raees Ahmed

Reviewer #2: Yes: Adnan Akhter

Reviewer #3: No

PLoS One. 2021 Sep 30;16(9):e0257863. doi: 10.1371/journal.pone.0257863.r004

Author response to Decision Letter 1

10 Sep 2021

Responses to the Editor and Reviewers’ comments

Additional Editor Comments (if provided):

The revised manuscript has been corrected by professional editing company.

In abstract, background of research and aim of the study are missing, please revise abstract to make it more focused.

Made changes according to the suggestions

What is NZ in abstract, this is a wrong way of presentation please revise, do you mean Burkholderia contaminans, if it is, then it is not properly cited.

Changed all instances of NZ to B. contaminans NZ

Page 2 lines 34 compounds or molecules not substances, and overall the MS need thorough English revision failing which MS will be not accepted.

Changed according to the suggestions

Overall, abstract is very poorly written, please revise it to make more focused.

Revised according to the suggestions

Page 3 line 65 line 85 species not italic correct in whole MS

Revised accordingly

Line 77 space

Revised accordingly

Line 92 to 101 it should described objective of your research and why it needed and then what are you presenting in this paper.

Revised accordingly

Did you check pathogenicity of Macrophomina phaseolina before conducting experiments?

We have checked the pathogenicity of Macrophomina phaseolina on jute seedlings and found jute seedlings dies after few days of infection, and turns brown. This result has been included in our recent paper by Zaman NR et al. (2020)

Ref:

1. Zaman NR, Kumar B, Nasrin Z, Islam MR, Maiti TK, Khan H. (2020) Proteome Analyses Reveal Macrophomina phaseolina's Survival Tools When Challenged by Burkholderia contaminans NZ. ACS Omega. 5(3):1352-1362. doi:10.1021/acsomega.9b01870

Line 120 it was not grown in shaker, you incubated in incubator shaker, how long old culture you used for DNA isolation

The statement was corrected according to the suggestion. It is mentioned in the same line that the bacterium was grown overnight and DNA was isolated from that culture.

The average shoot length, average root length and average height from the three repeated experiment was taken for statistical analysis shown in supplementary table 1.

Petri dish, P should be capital

Revised accordingly

Remove statistical part from each section and put it in the last of material and method in the single section describing the details how you analyzed the data as I mentioned.

Revised accordingly

Line 259 ascomycota group of fungi

Revised accordingly

Line 272 (P<0.05)

Revised accordingly throughout the MS

Table1 I think name of genes should be italic, follow in all tables

Revised accordingly

Coulumn one Fungal Species

Revised accordingly

Line 342 full name Macrophomina phaseolina should start follow in whole in whole MS same trend

Revised accordingly

Table 4 can be given as supplementary table

Revised accordingly

Line 441 strains

Revised accordingly

Conclusion should be short and easily understandable

Changed accordingly

Some of the references not follow the journal trends.

Changed accordingly

Attachment

Submitted filename: Response to the Editors Comments.docx

Click here for additional data file.^{(20.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0257863.r005

Decision Letter 2

Abhay K Pandey

14 Sep 2021

Plant growth promoting endophyte Burkholderia contaminans NZ antagonizes phytopathogen Macrophomina phaseolina through melanin synthesis and pyrrolnitrin inhibition

PONE-D-21-18655R2

Dear Dr. Islam,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Abhay K. Pandey

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

authors addressed all comments

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0257863.r006

Acceptance letter

Abhay K Pandey

22 Sep 2021

PONE-D-21-18655R2

Plant growth promoting endophyte Burkholderia contaminans NZ antagonizes phytopathogen Macrophomina phaseolina through melanin synthesis and pyrrolnitrin inhibition

Dear Dr. Islam:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Abhay K. Pandey

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Proposed melanin pathways in M. phaseolina based on homology search.

(TIF)

Click here for additional data file.^{(475.1KB, tif)}

S2 Fig

Qualitative assay for (a) siderophore, (b) ACC deaminase, and (c) nitrogen of B. contaminans NZ.

(TIF)

Click here for additional data file.^{(112.9KB, tif)}

S1 Table. Average root length, shoot length, and plant height of bacteria treated jute seedlings vs. untreated control in a pot experiment at 4, 7, and 10 days.

The data of three replicates per experiment are presented as means and standard deviations.

(DOCX)

Click here for additional data file.^{(17KB, docx)}

S2 Table. Fresh weight and dry weight of the bacteria treated jute seedlings vs. untreated control in a pot experiment at 4, 7, and 10 days.

The data of three replicates per experiment are presented as mean(s) and standard deviation(s).

(DOCX)

Click here for additional data file.^{(15.4KB, docx)}

S3 Table. Genomic features of Burkholderia contaminans NZ.

(DOCX)

Click here for additional data file.^{(14.1KB, docx)}

S4 Table. Location of genes involved in plant growth promotion activity detected from RAST, antiSMASH, and PIFAR analysis of the whole genome of B. contaminans NZ.

(DOCX)

Click here for additional data file.^{(19.1KB, docx)}

S5 Table. In vitro plant growth promotion attributes of B. contaminans NZ.

(DOCX)

Click here for additional data file.^{(17.3KB, docx)}

Attachment

Submitted filename: APAP.docx

Click here for additional data file.^{(16.9KB, docx)}

Attachment

Submitted filename: Responses to the reviewers comment.docx

Click here for additional data file.^{(34.1KB, docx)}

Attachment

Submitted filename: Response to the Editors Comments.docx

Click here for additional data file.^{(20.5KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Plant growth promoting endophyte Burkholderia contaminans NZ antagonizes phytopathogen Macrophomina phaseolina through melanin synthesis and pyrrolnitrin inhibition

Nazia R Zaman

Umar F Chowdhury

Rifath N Reza

Farhana T Chowdhury

Mrinmoy Sarker

Muhammad M Hossain

Md Ahedul Akbor

Al Amin

Mohammad Riazul Islam

Haseena Khan

Roles

Abstract

Introduction

Materials and methods

Bacterial, fungal strains and plant materials

Whole genome sequencing of B. contaminans NZ

Genome sequence analysis and annotation

Examining the presence of proteins fostering virulence and pathogenicity

Data deposition

In vivo growth promotion study of B. contaminans NZ on jute

Inoculation of jute seeds with the endophyte

In vivo pot experiment in hydroponic culture

Antifungal activity assay

Dual culture assay

Co-culture of M. phaseolina and B. contaminans NZ

Extraction of organic compounds from culture media

GC-MS analysis of ethyl acetate extract

HPLC and LC-MS analysis of methanolic extract

Identification of homologous melanin pathway(s) in M. phaseolina

Statistical analysis

Results

Pot tests for in vivo assay of plant growth promotion

Fig 1. In vivo effect of plant growth promoting endophytic bacteria B. contaminans NZ.

Whole genome data analysis

Table 1. List of genes involved in plant growth promotion activity detected from RAST, antiSMASH, PIFAR analysis of the B. contaminans NZ whole genome.

Most virulence genes absent in B. contaminans NZ

Table 2. List of major virulence related genes present or absent in B. contaminans NZ.

Antifungal activity assay

Fig 2. Antagonistic properties of B. contaminans NZ against six other phytopathogenic fungi.

Table 3. Growth inhibition induced by B. contaminans NZ on different plant pathogenic fungi.

Growth suppression and chromogenic aberration in B. contaminans NZ challenged M. phaseolina

Fig 3. Chromogenic aberration in B. contaminans NZ challenged M. phaseolina.

Chemical analysis of bio-active compounds using GC-MS

Table 4. List of potential compounds with their biological activities and retention times found in GC-MS analysis of B. contaminans NZ (B), M. phaseolina (M), and their co-culture (C).

Fig 4. Peaks from GC-MS analyses with potent volatile compounds.

Identification of pyrrolnitrin in methanolic extract of B. contaminans NZ culture

Fig 5. Identification of bioactive compounds in methanolic extract of B. contaminans NZ culture.

Identification of putative melanin pathways in M. phaseolina through homology-based search

Table 5. Homologous proteins of melanin pathways found in M. phaseolina.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Abhay K Pandey

Roles

Author response to Decision Letter 0

Decision Letter 1

Abhay K Pandey

Roles

Author response to Decision Letter 1

Decision Letter 2

Abhay K Pandey

Roles

Acceptance letter

Abhay K Pandey

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