suppression of fusarium wilt of banana with application of bio-organic fertilizers

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Pedosphere 24(5): 613–624, 2014 ISSN 1002-0160/CN 32-1315/P c 2014 Soil Science Society of China Published by Elsevier B.V. and Science Press Suppression of Fusarium Wilt of Banana with Application of Bio-Organic Fertilizers 1 ZHANG Nan 1 , HE Xin 1 , ZHANG Juan 2 , W. RAZA 1 , YANG Xing-Ming 1 , RUAN Yun-Ze 3 , SHEN Qi-Rong 1 and HUANG Qi-Wei 1,2 1 National Engineering Research Center for Organic-Based Fertilizers, Nanjing Agricultural University, Nanjing 210095 (China) 2 Shandong Product Quality Inspection Research Institute, Jinan 250102 (China) 3 College of Agronomy, Hainan University, Danzhou 571737 (China) (Received September 10, 2013; revised May 22, 2014) ABSTRACT Fusarium wilt is one of the most serious diseases of banana plants caused by soil-borne pathogen Fusarium oxysporum f.sp. cubense (FOC). In this study a pot experiment was conducted to evaluate the effects of different bio-organic fertilizers (BIOs) on Fusarium wilt of banana, including the investigations of disease incidence, chitinase and β-1,3-glucanase activities of banana plants, and FOC populations as well as soil rhizosphere microbial community. Five fertilization treatments were considered, including chemical fertilizer containing the same N, P and K concentrations as the BIO (control), and matured compost mixed with antagonists Paenibacillus polymyxa SQR-21 and Trichoderma harzianum T37 (BIO1), Bacillus amyloliquefaciens N6 (BIO2), Bacillus subtilis N11 (BIO3), and the combination of N6 and N11 (BIO4). The results indicated that the application of BIOs significantly decreased the incidence rate of Fusarium wilt by up to 80% compared with the control. BIOs also significantly promoted plant growth, and increased chitinase and β-1,3-glucanase activities by 55%–65% and 17.3%–120.1%, respectively, in the banana roots. The population of FOC in the rhizosphere soil was decreased significantly to about 10 4 colony forming units g 1 with treatment of BIOs. Serial dilution plating and denaturing gradient gel electrophoresis analysis revealed that the application of BIOs increased the densities of bacteria and actinomycetes but decreased the number of fungi in the rhizosphere soil. In general, the application of BIOs revealed a great potential for the control of Fusarium wilt disease of banana plants. Key Words: biocontrol, denaturing gradient gel electrophoresis, fungal disease, manure compost Citation: Zhang, N., He, X., Zhang, J., Raza, W., Yang, X. M., Ruan, Y. Z., Shen, Q. R. and Huang, Q. W. 2014. Suppression of Fusarium wilt of banana with application of bio-organic fertilizers. Pedosphere. 24(5): 613–624. INTRODUCTION Fusarium wilt of banana (Musa spp.), commonly known as Panama disease caused by Fusarium oxyspo- rum f.sp. cubense (E.F. Smith) Snyder & Hansen (Sny- der and Hansen, 1940), is one of the most serious fun- gal diseases and the major limiting factor for banana production worldwide (Lin et al., 2009). There are no effective chemical control measures for panama disease and the currently practiced corm injection procedure with fungicide carbendazim is tedious and environmen- tally unfriendly (Getha and Vikineswary, 2002). Other control methods such as field sanitation, soil treat- ments with fumigants, flood fallowing and crop rota- tion with nonhosts of the fungus have rarely provided long-term control in any production area (Ploetz et al., 1990). Currently, the selection of resistant cultivars is generally accepted as a disease control method and se- veral resistant cultivars (e.g., Farmosona) are grown in larger areas in Taiwan for the management of Fusa- rium wilt (Ploetz, 1990). As an alternative approach, the effectiveness of some plant growth-promoting and antagonistic bacteria against soil-borne pathogens has been widely evaluated (Saravanan et al., 2003). Un- fortunately, directly inoculation of biocontrol agents into soil may lead to poor activity of these microbes (Alabouvette et al., 2006). To overcome this problem, some integrated approaches, including enhancement of the activity of biocontrol agents by adding organic amendments, were attempted to achieve better effects (Saravanan et al., 2003). Hoitink et al. (1975) first put forward that com- post can be used as a peat substitute to control root pathogens. Later, the biocontrol research was increa- singly focused on developing the right combination of compost and antagonistic microbes. Trichoderma as- 1 Supported by the National High Technology Research and Development Program (863 Program) of China (No.2010AA10Z401). 2 Corresponding author. E-mail: [email protected].

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Page 1: Suppression of Fusarium Wilt of Banana with Application of Bio-Organic Fertilizers

Pedosphere 24(5): 613–624, 2014

ISSN 1002-0160/CN 32-1315/P

c© 2014 Soil Science Society of China

Published by Elsevier B.V. and Science Press

Suppression of Fusarium Wilt of Banana with Application of

Bio-Organic Fertilizers∗1

ZHANG Nan1, HE Xin1, ZHANG Juan2, W. RAZA1, YANG Xing-Ming1, RUAN Yun-Ze3, SHEN Qi-Rong1 andHUANG Qi-Wei1,∗2

1National Engineering Research Center for Organic-Based Fertilizers, Nanjing Agricultural University, Nanjing 210095 (China)2Shandong Product Quality Inspection Research Institute, Jinan 250102 (China)3College of Agronomy, Hainan University, Danzhou 571737 (China)

(Received September 10, 2013; revised May 22, 2014)

ABSTRACTFusarium wilt is one of the most serious diseases of banana plants caused by soil-borne pathogen Fusarium oxysporum f.sp. cubense

(FOC). In this study a pot experiment was conducted to evaluate the effects of different bio-organic fertilizers (BIOs) on Fusarium

wilt of banana, including the investigations of disease incidence, chitinase and β-1,3-glucanase activities of banana plants, and FOC

populations as well as soil rhizosphere microbial community. Five fertilization treatments were considered, including chemical fertilizer

containing the same N, P and K concentrations as the BIO (control), and matured compost mixed with antagonists Paenibacillus

polymyxa SQR-21 and Trichoderma harzianum T37 (BIO1), Bacillus amyloliquefaciens N6 (BIO2), Bacillus subtilis N11 (BIO3), and

the combination of N6 and N11 (BIO4). The results indicated that the application of BIOs significantly decreased the incidence rate

of Fusarium wilt by up to 80% compared with the control. BIOs also significantly promoted plant growth, and increased chitinase and

β-1,3-glucanase activities by 55%–65% and 17.3%–120.1%, respectively, in the banana roots. The population of FOC in the rhizosphere

soil was decreased significantly to about 104 colony forming units g−1 with treatment of BIOs. Serial dilution plating and denaturing

gradient gel electrophoresis analysis revealed that the application of BIOs increased the densities of bacteria and actinomycetes but

decreased the number of fungi in the rhizosphere soil. In general, the application of BIOs revealed a great potential for the control of

Fusarium wilt disease of banana plants.

Key Words: biocontrol, denaturing gradient gel electrophoresis, fungal disease, manure compost

Citation: Zhang, N., He, X., Zhang, J., Raza, W., Yang, X. M., Ruan, Y. Z., Shen, Q. R. and Huang, Q. W. 2014. Suppression of

Fusarium wilt of banana with application of bio-organic fertilizers. Pedosphere. 24(5): 613–624.

INTRODUCTION

Fusarium wilt of banana (Musa spp.), commonlyknown as Panama disease caused by Fusarium oxyspo-rum f.sp. cubense (E.F. Smith) Snyder & Hansen (Sny-der and Hansen, 1940), is one of the most serious fun-gal diseases and the major limiting factor for bananaproduction worldwide (Lin et al., 2009). There are noeffective chemical control measures for panama diseaseand the currently practiced corm injection procedurewith fungicide carbendazim is tedious and environmen-tally unfriendly (Getha and Vikineswary, 2002). Othercontrol methods such as field sanitation, soil treat-ments with fumigants, flood fallowing and crop rota-tion with nonhosts of the fungus have rarely providedlong-term control in any production area (Ploetz et al.,1990). Currently, the selection of resistant cultivars isgenerally accepted as a disease control method and se-

veral resistant cultivars (e.g., Farmosona) are grownin larger areas in Taiwan for the management of Fusa-rium wilt (Ploetz, 1990). As an alternative approach,the effectiveness of some plant growth-promoting andantagonistic bacteria against soil-borne pathogens hasbeen widely evaluated (Saravanan et al., 2003). Un-fortunately, directly inoculation of biocontrol agentsinto soil may lead to poor activity of these microbes(Alabouvette et al., 2006). To overcome this problem,some integrated approaches, including enhancement ofthe activity of biocontrol agents by adding organicamendments, were attempted to achieve better effects(Saravanan et al., 2003).

Hoitink et al. (1975) first put forward that com-post can be used as a peat substitute to control rootpathogens. Later, the biocontrol research was increa-singly focused on developing the right combination ofcompost and antagonistic microbes. Trichoderma as-

∗1Supported by the National High Technology Research and Development Program (863 Program) of China (No. 2010AA10Z401).∗2Corresponding author. E-mail: [email protected].

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614 N. ZHANG et al.

perellum and sewage sludge compost were utilized tosuppress Fusarium wilt of tomato (Cotxarrera et al.,2002). Trillas et al. (2006) also reported the significantbiocontrol of rhizoctonia disease of cucumber by theapplication of a mixture of agricultural compost andTrichoderma spp. It was thought that compost can pro-vide nutrients for microbes, thus increasing the anta-gonist’s viability and facilitating their survival in rhi-zosphere and colonization on plant roots (Boehm etal., 1993).

Bacillus species have been successfully applied forthe biocontrol of soil-borne pathogens (Ongena andJacques, 2008). Paenibacillus polymyxa has been foundnot only to be a plant growth-promoting rhizobacte-ria, but also to be an effective biocontrol agent, whichcan produce a broad range of peptide metabolites withantibacterial and/or antifungal activities (Raza et al.,2008) and form highly resistant endospores to bothchemical and physical stresses. T. harzianum has alsobeen used as an antagonistic fungal strain with rea-sonable effects against soil-borne pathogens (Wu etal., 2009). A novel bio-organic fertilizer (BIO) by fer-menting matured compost with biocontrol strains P.polymyxa SQR-21 and T. harzianum T37 was deve-loped by our laboratory. This BIO was previouslyshown to suppress Fusarium wilt disease, and promotethe growth of watermelon plants in greenhouse andfield experiments (Wu et al., 2009). Two other an-tagonistic strains, Bacillus amyloliquefaciens N6 andBacillus subtilis N11, were also isolated from the ba-nana continuously cropping soils and were fermentedwith matured compost to get specific BIOs for bananaplants. In the present study, therefore, a pot experi-ment was carried out to assess the effects of the com-binations of organic fertilizer and these antagonisticmicroorganisms on Fusarium wilt of banana, includingthe investigations of disease incidence, chitinase andβ-1,3-glucanase activities of banana plants, and po-pulations of FOC as well as soil rhizosphere microbialcommunity.

MATERIALS AND METHODS

Bacterial and fungi strains

The pathogen fungal strain Fusarium oxysporumf.sp. cubense (FOC), as well as a tested antagonisticbacterial strain P. polymyxa SQR-21 and a tested an-tagonistic fungal strain T. harzianum T37, were pro-vided by the Jiangsu Provincial Key Lab for OrganicSolid Waste Utilization, Nanjing Agriculture Univer-sity, China (Wu et al., 2009; Ling et al., 2010). Twobacterial isolates showing the strongest antagonism

against FOC (data not shown) were selected as an-tagonistic agents to fortify organic fertilizer. The 16SrRNA gene sequences of one bacterial isolate, desig-nated as N6, showed 100% identity to B. amyloliquefa-ciens strain 16. The other isolate, designated as N11,shows 100% identity to B. subtilis strain DYJL26. Se-quences of both strains were deposited into GenBankunder accession Nos. GQ452909 and GQ452910, re-spectively. The bacterial strains were stored in Luria-Bertani medium at −80 ◦C in 20% glycerol while thefungal strain was maintained on potato dextrose agarplates and stored at 4 ◦C.

BIO preparation

Organic fertilizer used for BIOs preparation wascomposed of amino acid fertilizer and pig manure com-post (1:1). Amino acid fertilizer was made from oilrapeseed cakes that were hydrolyzed by microbial en-zymes for 7 d (Zhang, S. S. et al., 2008). This aminoacid fertilizer contained 442 g kg−1 organic matter,129 g kg−1 sum of amino acids, small molecular pep-tides and oligo peptides, 44 g kg−1 nitrogen (N), 35 gkg−1 P2O5, and 7 g kg−1 K2O. Pig manure compostwas made by Tianniang Limited, China by compostingpig manure at a temperature range of 30–70 ◦C for 25d. This compost was composed of 304 g kg−1 organicmatter, 20 g kg−1 N, 37 g kg−1 P2O5, and 11 g kg−1

K2O.For the BIOs preparation, 1 000 mL suspensions of

the mixture of SQR-21 (1 × 109 colony forming units,CFU, mL−1) and T37 (1 × 106 CFU mL−1), N6 or/andN11 (1 × 109 CFU mL−1) and organic fertilizer (5 kg)were thoroughly mixed in a 500 mm × 360 mm × 175mm plastic case for secondary fermentation. The mix-ture was maintained at 40%–45% moisture under roomtemperature (20–31 ◦C) for 6 d and manually invertedevery day. Then, the mixture was spread for air-dryingin a ventilation room at room temperature for 2 d un-til water contents were less than 30%. During the se-condary fermenting, temperature and bacterial densi-ty of the substrate were observed daily. The compostscontaining bacterial populations higher than 1 × 109

CFU g−1 DW or fungal populations higher than 1 ×105 CFU g−1 DW were defined as a BIO. The BIO wasstored at 4 ◦C prior to use in the experiment.

Pot experiment

The properties of the soil used for pot experimentwere listed as follows: pH of 5.4, organic matter of 7.3g kg−1, available N of 79 mg kg−1, available P of 31 mgkg−1 and available K of 40 mg kg−1. The soil was pre-inoculated with the spore suspension of FOC to obtain

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SUPPRESSION OF BANANA WILT WITH BIO-FERTILIZER 615

the final concentration of 1 × 105 CFU g−1. Bananaseedlings were grown in nursery and after 20 d, theseedlings with 3–4 true leaves were transplanted intothe pots with 10 kg soil. Five fertilization treatmentswere designed as follows: chemical fertilizer containingthe same NPK concentrations as the BIO (control, T1),and matured compost mixed with the combination ofSQR-21 and T37 (BIO1, T2), N6 (BIO2, T3), N11(BIO3, T4), and the mixture of N6 and N11 (BIO4,T5). Nursery and pot soils were supplemented with20 and 5 g kg−1 fertilizer in each treatment, respec-tively. Each treatment has three blocks, and each blockcomprised 10 pots in completely random design. Theseedlings were grown in the greenhouse under naturalconditions (23–30 ◦C).

Seedling infection by F. oxysporum was recordedevery day and the cumulative numbers of the infectedplants were recorded from the day of transplantationuntil 56 d. Disease incidence was calculated as the per-centage of diseased plants over the total number ofgrowing plants in each block. In addition, three plantswere randomly sampled from each treatment at harvesttime, and their dry weights, heights and stem girthswere measured.

Chitinase and β-1,3-glucanase activity determination

At harvest time, three plants were randomly sam-pled from each treatment. Samples (500 mg) of leavesand roots were separately ground to a fine powder in amortar under liquid nitrogen. One portion of the pow-der was suspended in 5 mL of 50 mmol L−1 potas-sium phosphate buffer (pH 7.8), homogenized and cen-trifuged at 14 000 × g for 15 min at 4 ◦C. For chitinaseactivity determination, the supernatant was dilutedtwo times. The colloid chitin was prepared accordingto Wu et al. (2009). The chitinase activity was de-termined based on the method described by Schraud-ner et al. (1992) using N-acetyl-amino-glucosamine asstandard. One unit of chitinase activity was definedas the amount of enzyme that produced 1 μg of N-acetyl-amino-glucosamine per hour from chitin underthese assay conditions. The activity of β-1,3-glucanasewas determined using the method of Schraudner et al.(1992) using laminarine as standard. One unit of β-1,3-glucanase activity was defined as the amount of en-zyme that produced 1 nmol reduced sugar per secondunder the assay conditions.

Recovery of FOC from soil

Culture method. Banana roots were carefullyseparated from the soil and lightly shaken to removeloosely attached soil as bulk soil. The roots with rhi-

zosphere soil were cut into pieces of about 20 mm.Ten grams of bulk soil or the mixture of root piecesand rhizosphere soil were separately placed in 90 mLsterile distilled water, shaken for 30 min and thenkept for 10 min. The number of FOC CFU in thebulk and rhizosphere soils was determined by serialdilutions on Komada’s medium (Komada, 1975). Thestrains belonging to F. oxysporum were identified bytheir morphological characteristics (Komada, 1975)and counted.

Real-time polymerase chain reaction. DNA wasextracted from the bulk and rhizosphere soils using theUltraClean soil DNA isolation kit (Mo Bio, USA) ac-cording to the manufacture’s instructions. The primersreported by Lievens et al. (2005) were used for thedetection of pathogen. A 377-bp fragment of internaltranscribed spacer (ITS) region of FOC was amplifiedand cloned in PMD 18-T vector. The concentration wasadjusted to the number of 1 × 1011 ITS copies g−1, andthe standard was diluted step by step by 10-fold to ob-tain the standard curves. Real-time polymerase chainreaction (PCR) amplifications were performed in a to-tal volume of 50 μL using a SYBR@ Premix Ex TaqTM

(Takara, China). The reaction mixtures contained a fi-nal concentration of 25 μL SYBR@ Premix Ex TaqTM,1 μL each primer (10 μmol L−1), 1 μL ROX referencedye II (50 ×), 2 μL template DNA and 20 μL sterilewater. The thermal cycling conditions were: 95 ◦C for30 s, followed by 40 cycles of 95 ◦C for 5 s, 60 ◦C for34 s, and 72 ◦C for 15 s, and finally a dissociation stage(95 ◦C for 15 s, 60 ◦C for 60 s, and 95 ◦C for 15 s).The amplification results were analyzed with 7500 FastSystem SDS software (ABI, USA).

Determination of soil microbe populations

Microbial populations in the rhizosphere soil weredetermined at 56 d after transplantation. The CFUcounts were made by serial dilutions of the rhizospheresoil suspension on appropriate mediums: beef extractmedium for bacteria (beef extract 5 g, peptone 10g, NaCl 5 g, H2O 1000 mL, pH 7.2), Gause’s No. 1medium for actinomycetes (amidulin 2 g, KNO3 0.1g, K2HPO4 0.05 g, MgSO4·7H2O 0.05 g, NaCl 0.05g, FeSO4·7H2O 0.001 g, H2O 100 mL, pH 7.2), andMartin’s rose bengal medium for fungi (KH2PO4 1 g,MgSO4·7H2O 0.5 g, peptone 5 g, glucose 10 g, H2O1000 mL, pH 7.0, 1% rose bengal solution 3.3 mL, and1% streptomycin stock solution were added after ste-rilization) (Huang et al., 2006). Plates were incubatedin dark at 28 ◦C for fungi and 30 ◦C for bacteria andactinomycetes. The populations of bacteria and acti-nomycetes were counted after 2–4 d and fungi after

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616 N. ZHANG et al.

5–7 d of incubation. Four replicates were used for eachtreatment.

Denaturing gradient gel electrophoresis (DGGE)

PCR conditions. DNA was extracted as de-scribed above and microbial diversity of the sam-ples was determined by using of PCR-DGGE (Luoet al., 2010). Briefly, a 233-bp fragment including a40-bp GC-clamp of bacterial DNA was amplified usi-ng the universal bacterial primers 341f + GC (5′-GCclamp-TACGGGAGGCAGCAG-3′) and 518r (5′-GCclamp-ATTACCGCGGCTGCTGG-3′) (Muyzer et al.,1993). PCR was performed using 2.5 μL 10 × Ex Taqbuffer (20 mmol L−1 Mg2+, Takara, China), 1 μL 2.5mmol L−1 dNTP mixture, 0.3 μL 5 units μL−1 Ex Taqpolymerase (Takara), 1 μL of each primer (10 pmolμL−1), 1 μL diluted template and H2O in the total vo-lume of 25 μL. Bacterial PCR was performed using thefollowing cycle conditions: 94 ◦C for 5 min, followed by30 cycles of 94 ◦C for 30 s, 61 ◦C for 30 s and 72 ◦C for15 s, and then 72 ◦C for 10 min. For the fungi, the pri-mer pair NS1 (5′-GTAGTCATATGCTTGTCTC-3′)and the fungus-special primer GC fungi (5′-GC clamp-ATTCCCCGTTACCCGTTG-3′) (Das et al., 2007)were used to amplify a 370-bp fragment, including a40-bp GC-clamp. Cycle conditions in the fungal PCRwere as follows: 94 ◦C for 5 min, followed by 30 cyclesof 94 ◦C for 30 s, 58 ◦C for 30 s and 72 ◦C for 25 s,and 72 ◦C for 10 min. The amount of DNA in eachsample was estimated by image analysis using Gene-Tools (SynGene, UK) on digital images of the agarosegels obtained with GeneSnap (SynGene) to ensure thatequal amounts of DNA from the samples were loadedonto the DGGE gel.

DGGE condition. The DGGE was performedusing the D-GENE System (Bio-Rad, USA) DGGEequipment. Equal amounts of DNA were loaded onto8% (w/v) polyacrylamide gels (40% acrylamide/bis-solution, 37.5:1) with denaturing gradients ranging40%–60% for the bacterial DNA and 25%–40% for thefungal DNA. The gels were run at 60 ◦C and 80 Vfor 16 h. The digital images of the gels were analyzedusing image analysis, thereby quantifying the bands(Quantity One 4.6.3, Bio-Rad). The relative intensityof a band was expressed as the ratio between the in-tensity of that band and the total intensity of all bandsin that lane.

DNA sequencing. The DGGE bands were ex-cised and the DNA from each band was eluted in 50μL of sterile water, overnight at 4 ◦C. One μL of eacheluted DNA band was re-amplified using the condi-tions described above. PCR products were analyzed

by DGGE to confirm that the expected products havebeen isolated. The samples yielding a single band co-migrating with the original sample were then excisedand amplified with the primers without GC-clamp,purified and sent to GenScript Company (Nanjing,China) for sequencing. The sequences of bacteria andfungi gene fragments were searched with basic localalignment search tool in GenBank to find the closestknown relatives of the partial bacteria and fungus se-quences.

Statistical analysis

Differences among the treatments were calculatedand statistically analyzed by using analysis of variance(ANOVA) and Duncan’s multiple range test at P <

0.05. The SPSS version 17.0 (SPSS Inc., Chicago, IL)was used for statistical analysis. Cluster analysis wasperformed with the UPGMA algorithm to study ge-neral patterns of community similarity in the QuantityOne (Quantity One 4.6.3, Bio-Rad) (P < 0.05). Thetree was constructed by the software MEGA 4 withbootstrapping value (range 0–100).

RESULTS

Disease incidence of Fusarium wilt

The application of BIOs all significantly inhibitedthe Fusarium wilt of banana in the pot experimentwhen compared with the control (Fig. 1, F = 102.3,

Fig. 1 Effects of different fertilization treatments on Fusarium

wilt incidence of banana at 56 d after transplantation. Bars

with the same letter are not significantly different at P < 0.05

using Duncan’s multiple range test. T1 = chemical fertilizer

containing the same N, P and K concentrations as bio-organic

fertilizer (BIO) (control); T2 = matured compost mixed with

the combination of Paenibacillus polymyxa SQR-21 and Tricho-

derma harzianum T37 (BIO1); T3 = matured compost mixed

with Bacillus amyloliquefaciens N6 (BIO2); T4 = matured com-

post mixed with Bacillus subtilis N11 (BIO3); T5 = matured

compost mixed with N6 and N11 (BIO4).

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SUPPRESSION OF BANANA WILT WITH BIO-FERTILIZER 617

df = 4, 10, P = 0.0001). After 56 d of transplanta-tion, the disease incidence rate of 93% was determinedin the control treatment. The incidence rate in all theBIO treatments was lower than 35%, while the lowestdisease incidence rate of 14% was detected in T3.

Growth assessment of banana plants

The application of BIOs not only suppressed theFusarium wilt but also significantly promoted thegrowth of banana plants, indicated by the increase inheights, stem girths and dry weights of the bananaplants as compared with the control (Table I). Theheight and stem girth of plants were increased by73.6%–108.6% and 60.1%–98.7%, respectively, com-pared with the control (F = 83.322, df = 4, 10, P =0.0001 for height; F = 23.327, df = 4, 10, P = 0.0001for stem girth). The dry weights of plants were in-creased by 861%–1 710% compared with the control(F =17.147, df =4, 10, P =0.0001). The best growthstatus was obtained in T3.

TABLE I

Effects of different fertilization treatments on the growth of ba-

nana plants at 56 d after transplantation

Treatmenta) Plant height Stem girth Dry weight

cm g plant−1

T1 36.0±0.7b)cc) 6.2±0.2c 1.0±0.3c

T2 62.5±2.1b 10.0±0.7b 9.6±2.4b

T3 81.5±3.7a 12.4±1.3a 18.1±4.2a

T4 68.3±3.7b 11.5±1.3a 14.3±4.2a

T5 75.1±4.7a 12.1±0.3a 17.9±1.8a

a)See Fig. 1 for the descriptions of abbreviations.b)Means±standard errors (n = 3).c)Means followed by the same letter within each column are not

significantly different at P < 0.05 using Duncan’s multiple range

test.

Chitinase and β-1,3-glucanase activities

The chitinase activity in banana leaves was ge-nerally similar among the five treatments (F = 6.52,df = 4, 10, P = 0.008), only T3 and T4 treatments ledto a significant activity increase when compared withthe control (Fig. 2). While all the BIO treatments ledto a significant increase of the enzyme activity in theroots compare with the control (F = 12.999, df = 4,10, P = 0.001). The highest chitinase activity in theroots was found in the T3 treatment, which was 14.5%higher than that in the control.

The β-1,3-glucanase activates in banana leaves ofthe T2, T3 and T5 treatments were significantly in-creased as compared with the control (Fig. 2). Howe-ver, the highest β-1,3-glucanase activity was recordedin T3, showing a 120.1% increase relative to the con-trol (F = 30.168, df = 4, 10, P = 0.0001). In the roottissues, the β-1,3-glucanase activities were increasedby 11.7% and 14.1%, respectively, in the T2 and T3treatments as compared with the control (F = 2.018,df = 4, 10, P = 0.168), while the β-1,3-glucanase ac-tivities in the T4 and T5 treatments were not signifi-cantly different from those in the T2, T3 and controltreatments.

Recovery of FOC from soil

Culture method. The FOC was recovered fromthe bulk and rhizosphere soils. In the bulk soil, onlythe FOC population in T4 was significant lower thanthat in the control, while the pathogen density wassimilar among all the other four treatments (Fig. 3a,F = 2.601, df = 4, 10, P = 0.100). On the other hand,there was a significant difference in CFU levels of FOCin the rhizosphere soil between the treatments with or

Fig. 2 Effects of different fertilization treatments on chitinase and β-1,3-glucanase activities in leaves and roots of banana plants.

Bars with the same letter(s) for each plant organ are not significantly different at P < 0.05 using Duncan’s multiple range test. See

Fig. 1 for the descriptions of the abbreviations of T1, T2, T3, T4 and T5. U = unit; FW = fresh weight.

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618 N. ZHANG et al.

Fig. 3 Recovery of Fusarium oxysporum with culture method (a) and real-time polymerase chain reaction (b) from the rhizosphere and

bulk soils of banana plants under different fertilization treatments. In the real-time polymerase chain reaction, only the relative copies

of the internal transcribed spacer region of F. oxysporum are shown. Bars with the same letter(s) for each soil are not significantly

different at P < 0.05 using Duncan’s multiple range test. See Fig. 1 for the descriptions of the abbreviations of T1, T2, T3, T4 and

T5. CFU = colony forming unit.

without the application of BIOs (F = 109.142, df = 4,10, P = 0.0001). When FOC populations were above 3× 104 CFU g−1 in the rhizosphere soil, the disease inci-dence rate was higher than 90% (Figs. 1 and 3). Supple-mentation with BOI1 significantly decreased the num-ber of FOC in the rhizosphere soil to 1 × 104 CFU g−1;while the application of the specific BIOs for banana,made by the combination of matured compost and theantagonist isolated from banana planted soil (named asBIO2–4), could further decrease the number of FOC tolower than 7 × 103 CFU g−1 in the rhizosphere soil.The lowest population of FOC (2 × 103 CFU g−1) wasobtained in the T3 treatment.

Real-time PCR. The linear equation betweenthe concentration of DNA template (x, logarithm ofthe copy number of templates per mL) and cyclethreshold value (Ct) was conducted as:

Ct = −3.57x + 27.62 (R2 = 0.999) (1)

Since no internal reference was taken in the real-time PCR, only the relative copy numbers of the ITSregion are shown in Fig. 3b. The results revealed a si-milar pattern with the culture method. No significantdifference of the pathogen population was found in thebulk soil among the treatments (F = 2.056, df = 4,10, P = 0.162). However, the copies of FOC in the rhi-zosphere soil treated with BIOs were significant lowerthan those in the control (F = 31.959, df = 4, 10,P = 0.0001). The FOC populations in the control was1.59 × 105 copies g−1, while amendment of BIOs de-creased the density of the pathogens to less than 6 ×104 copies g−1 in the rhizosphere soil.

Soil microbial diversity

The densities of cultivable bacteria, actinomycetesand fungi in the rhizosphere soil were monitored at56 d after transplantation. The application of BIOs si-gnificantly increased the population of actinomycetes(F = 103.96, df = 4, 10, P = 0.0001), while signifi-cantly decreased the fungal density in the rhizospheresoil (F = 111.503, df = 4, 10, P = 0.0001) (Ta-ble II). In addition, T3 and T4 treatments signifi-cantly increased the population of bacteria as com-pared with the control (Table II, F = 7.687, df = 4,10, P = 0.004). In relation to the control, the popu-lations of bacteria and actinomycetes were increased by38.7%–108.5% and 79.6%–338.4%, respectively, whilethe numbers of fungi were decreased by 38.5%–83.7%after application of BIOs. The T3 and

TABLE II

Densities of culturable bacteria, actinomycetes and fungi in the

rhizosphere soils of banana plants under different fertilization

treatments

Treatmenta) Bacteria Actinomycete Fungi

× 106 g−1 × 105 g−1 × 104 g−1

T1 47.0±1.0b)cc) 26.0±3.6d 124.7±11.6a

T2 66.3±6.4bc 46.7±5.7c 71.7±5.7b

T3 98.0±18.0a 59.3±4.2b 20.3±5.0d

T4 80.0±18.0ab 108.0±6.00a 33.3±1.5c

T5 65.7±3.2bc 114.0±10.8a 76.7±5.7b

a)See Fig. 1 for the descriptions of abbreviations.b)Means±standard errors (n = 3).c)Means followed by the same letter(s) within each column are

not significantly different at P < 0.05 using Duncan’s multiple

range test.

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SUPPRESSION OF BANANA WILT WITH BIO-FERTILIZER 619

T4 treatments especially decreased the fungal density.

DGGE

PCR-DGGE analysis of fungal communities in therhizosphere soil showed that there was no significantdissimilarities in species composition and abundance

of the fungal communities (number of occurring bands)(Fig. 4a). It seemed that the application of BIOs didnot significantly affect the diversity of the fungi species.For further information, 8 bands from the gel wereselected for sequencing (Fig. 4b) and the phylogenetictree of these sequences was constructed by MEGA 4

Fig. 4 Original denaturing gradient gel electrophoresis (DGGE) patterns (a) and dendrogram of DGGE profiles (b) of the fungal

community in the rhizosphere soil of banana plants under different fertilization treatments. Bands indicated by numbers 1 to 8 were

excised, and after re-amplification, subjected to sequencing. See Fig. 1 for the descriptions of the abbreviations of T1, T2, T3, T4 and

T5.

Fig. 5 Phylogenetic tree of the sequence from fungal denaturing gradient gel electrophoresis profiles. Bands 1 to 8 are indicated in

Fig. 4a.

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620 N. ZHANG et al.

(Fig. 5). The bands’ sequence identities were classi-fied as follows: band 1, uncultured Cystofilobasidiales(aff. Guehomyces); band 2, uncultured Tremellaceae;band 3, Fusarium oxysporum; band 4, uncultured Cha-etomium; band 5, Cryptococcus daszewskae; band 6,Cladosporium sp.; band 7, Trichoderma koningii; andband 8, Penidiella columbiana. Among them, band 3represented the FOC pathogen, but no significant dif-ference of the gray of the band was found among thetreatments. The band 7 represented a common fungalbiocontrol agent. Detail function of these fungal speciesor subspecies in soils needs to be further studied.

DGGE fingerprinting of bacterial diversity indi-cated that the application of BIOs, especially the spe-cific fertilizers BIO2–4, significantly increased bacterialspecies, which was in accordance with the plate coun-ting results. The UPGMA dendrogram revealed thatthe structure of bacterial community in T2 was moresimilar to that in T1, while T3, T4 and T5 were inthe same branch (Fig. 6a). For further information, 15bands from the gel were sequenced (Fig. 6b) and thephylogenetic trees of these sequences were constructedby MEGA 4 (Fig. 7). The identifications of these se-quences are listed as follows: band 1, Pseudomonassp.; bands 2 and 3, Pseudomonas fluorescens; bands4–6, Pseudomonas sp.; band 7, Flavobacteriaceae bac-terium; band 8, Paenibacillus polymyxa; band 9, Bacil-lus circulans; band 10, Pseudomonas sp.; bands 11and 12, Bacillus sp.; band 13, uncultured Xanthomon-adaceae bacterium; band 14, uncultured Verrucomicro-bia bacterium; and band 15, Rhodanobacter sp. The

tree indicated that Pseudomonas sp. was the main mi-crobial community in the rhizosphere. The band 8 rep-resented Paenibacillus polymyxa, and the bands 9, 11and 12 represented Bacillus sp. These identified strainswere similar to the inoculants used in BIOs, namelySQR-21, N6 and N11. The application of the specificfertilizer led to the appearance of bands 13–15, but thedetail functions of these microorganisms are remainedunknown.

DISCUSSION

In this study, we used a mixture of amino acidfertilizer and matured pig manure compost as amedium, to which the strains P. polymyxa SQR-21, T.harzianum T37 and two Bacillus strains N6 and N11were inoculated. The BIO significantly decreased theincidence rate of wilt disease by 64% to 86% comparedwith the control. The results were consistent with theobservations that the soil application of P. fluorescenswith neem cake gives the best results against the Fusa-rium wilt of banana (Saravanan et al., 2003). Our re-sults were also in agreement with Kay and Stewart(1994), who reported the significant biocontrol of whiterot disease of onion by the application of a mixture oforganic fertilizers and antagonists. The protection ofthe banana plants from FOC by the BIOs was also re-flected by the growth promotion of the plants.

Productions of pathogenesis-related proteins likechitinase and β-1,3-glucanase in host plant not onlyhydrolyze chitin and β-1,3-glucan of fungal cell wallsbut also release elicitors from the walls of fungi which

Fig. 6 Original denaturing gradient gel electrophoresis (DGGE) profiles (a) and dendrogram of DGGE profiles (b) of the bacterial

community in the rhizosphere soil of banana plants under different fertilization treatments. Bands indicated by numbers 1 to 15 were

excised, and after re-amplification, subjected to sequencing. See Fig. 1 for the descriptions of the abbreviations of T1, T2, T3, T4 and

T5.

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SUPPRESSION OF BANANA WILT WITH BIO-FERTILIZER 621

Fig. 7 Phylogenetic tree of the sequence from bacterial denaturing gradient gel electrophoresis profiles. Bands 1 to 15 are indicated

in Fig. 6a.

stimulate various defense responses in plants (Duttaet al., 2008). Usually, the beneficial microbes can in-duce the accumulation of such pathogenesis-relatedproteins in plant for against the pathogen, which iscalled induced systemic resistance (Pieterse and vanLoon, 1999). Our results showed that the BIOs, espe-cially BIO2 in the T3 treatment, significantly affectedthe activities of β-1,3-glucanase and chitinase in theroots and leaves of banana. These results are consistwith some previous studies (Dutta et al., 2008; Wuet al., 2009), which revealed that BIO effectively in-duces the activities of hydrolytic enzymes in the ba-nana plants, resulting an increased induced systemicresistance of the plant to the pathogen and thus lea-ding to the suppression of the Fusarium wilt. Though

the change of the activity did not seem drastic, thismight be one of the mechanisms by which BIOs pre-vented the banana plants from invading by FOC in thisstudy. In addition, some previous researchers have re-ported that the type and intensity of chitinase and β-1,3-glucanase isoforms are affected during the inducedsystem resistance (Roberti et al., 2008), so some fur-ther studies (e.g., isoelectric focusing) are required toreveal the detailed mechanisms.

The pathogen population in the soil was monitoredusing both plate counting and quantitative real-timePCR methods. Before the experiment, the control soilwas studied for the existence of FOC and the results in-dicated that the concentration of FOC was below 100CFU g−1, much lower than the density of FOC de-

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622 N. ZHANG et al.

tected in the inoculated soil. Therefore, the Komada’smedium was applied for the measurement of FOC inthe soil, and the primers for detection of all the FOCpublished by Lievens et al. (2005) were used in this ex-periment. Both methods revealed that the applicationof BIO significantly decreased the density of FOC inthe rhizosphere soil, while there was no remarkable dif-ference in the bulk soil. When FOC populations wereabove 3 × 104 CFU g−1 in the rhizosphere soil, thedisease incidence rate was higher than 90%. Supple-mentation with BIO decreased the FOC population inthe rhizosphere soil to 1 × 104 CFU g−1. Especially,the specific bio-organic fertilizer (BIO2–4) reduced thepopulation of the pathogen to less than 7 × 103 CFUg−1 in the rhizosphere soil. This phenomenon is also inaccordance with the results of the relationship betweenthe FOC spore concentration and disease severity men-tioned above.

The copy numbers of ITS region of fungi are knownto vary, which ranges from about 30 to 200 (Herrera etal., 2009). According to this scale, the real-time PCRresults are approximately in consistent with the cultureresults. Differences between PCR and culture methodmight be due to insufficient extraction of the soil DNAand homogenization of the samples, factors affectingthe growth of different isolates, or possibly, the pre-sence of antagonistic microbial species. Another ma-jor difference between PCR and culture is that PCRalso detects non-culturable fungi, which may explainin part the higher detection rate by PCR (Boutaga etal., 2003).

Soil microbial diversity is considered critical tomaintain soil health and quality, and different agri-cultural practices can influence soil microbial diver-sity and even the suppressive level of plant diseases(Garbeva et al., 2006; Bever et al., 2012; Lang etal., 2012). Large soil microbial biomass, high diver-sity, and dynamic balance of microbial composition arethought to be important for the formation and con-struction of suppressive soil (Weller et al., 2002). Thenative healthy soils usually hold diverse set of mi-crobes. Though some pathogens might be existed inthis system, the interactions between various microbeswill keep the balance of the system and none of thembecome dominant. While in a soil system with less mi-crobial diversity, the pathogens may become dominantin the rhizosphere because of the lack of other microbes(Badri and Vivanco, 2009). In this study, significantincreases of the populations of bacteria and actino-mycetes were found with BIO application. These re-sults were in accordance with Bottomley et al. (2006),Garbeva et al. (2006) and Liu et al. (2013), indicating

that BIOs application might suppress the soil-bornedisease by modulating the microbial community. Itshould be noticed that the correlation between shiftof microbial community (e.g., increase or decrease ofspecific population such as bacteria or fungi) and soilquality was variable, possibly depending on the climatecondition, soil properties, local microbial compositionand so on (Luo et al., 2010; Bever et al., 2012; Lang etal., 2012; Liu et al., 2013).

Culture-dependent techniques are restricted by thefact that more than 90% of the indigenous microor-ganisms are not readily cultivable using standard tech-niques. In order to reveal the detail community of mi-croorganisms, DGGE was carried out to investigatethe community of fungi and bacteria in the rhizospheresoils. Application of BIOs didn’t significantly changethe diversity and intensity of fungi in the rhizospheresoil, while a previous study (Russo et al., 2008) in-dicated that the fungal community structure changeswhen biocontrol agent Azospirillum brasilense is ap-plied. Scherwinski et al. (2007) reported that the intro-duction of antifungal strains into the strawberry rhizo-sphere causes only minor and transient effects on thecomposition of the rhizosphere microbiota. However,obvious differences of bacterial structure were foundamong the treatments and the amendment of BIOssignificantly increased the diversity of bacteria, espe-cially application of the specific fertilizer (BIO2–4).Adesina et al. (2009) reported that biological controlagent Pseudomonas jessenii RU47 alters the bacterialcommunity of the soil affected by Rhizoctonia solani.Zhang, B. G. et al. (2008) also demonstrated that ino-culation of Bacillus thuringiensis significantly changesthe phyllosphere bacterial community structure. Pre-vious report has highlighted that the diversity of bacte-rial community is a key factor affecting soil health andthe suppression of fungi pathogen (Ren et al., 2008).Therefore we suggested that the bacterial communitywas an important factor affecting Fusarium wilt. Itcould be speculated that the BIOs suppressed the dis-ease not only by reducing FOC population but also byincreasing soil fungistatic conditions, especially by in-creasing bacterial diversity and population of beneficialagents, which might restrain the pathogenicity and ac-tivity of FOC.

In addition, pre-experiments revealed that theBIOs could promote the growth of banana seedlings(data not shown). It should be noticed that the growthpromotion effect by BIOs was much less significantthan the biocontrol effect (plant height increased by23.2%–38.3% for promotion, compared with 73.6%–108.6% for the biocontrol; while dry weight increased

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SUPPRESSION OF BANANA WILT WITH BIO-FERTILIZER 623

by 149%–281% for promotion, compared with 861%–1 710% for biocontrol, data not shown). So the growthpromotion effect might be partially involved in the sup-pressive effect, but it was not the main contributor forsuccessful biocontrol.

Here we also found that the better biocontrol effectwas obtained in the treatments applied with N6 and/orN11. This suggested that we must specific antagonis-tic microbes in situ to control wilt of different plantspecies and suitable organic substrates were also veryimportant to get a good biological control. One reviewhas discussed this point from an evolutionary perspec-tive and provided information for managing disease-suppressive soils (Kinkel et al., 2011). These resultstogether with other reports (Zhang, S. S. et al., 2008;Ling et al., 2010; Ling et al., 2011; Luo et al., 2010;Yang et al., 2010; Zhao et al., 2010; Zhang et al., 2011)provide a breakthrough in the research work of biolo-gical control of soil born pathogen diseases. In addition,among these treatments, the best biocontrol effect wasobtained in the T3 treatment (N6 strain solely inocu-lated fertilizer), which is not consistent with one pre-vious study by Trillas et al. (2006), who indicated thata better biocontrol effect can be obtained by combi-nation of different antagonists. Different strains mightshow different colonization behaviors in the soil. Theinteraction among them is also very complex, and fur-ther study is needed to gain a deeper understanding.

CONCLUSIONS

Well-organized combinations of organic fertilizerwith different antagonistic strains and a reasonable ap-plication of BIO could significantly suppress the Fusa-rium wilt of banana. The possible protection mecha-nism involved in this product might be attributed toinduced accumulation of pathogenesis-related proteins(chitinase and β-1,3-glucanase) in banana roots, sup-pression of pathogen population, and regulation of soilmicrobial community for improving its fungistatic con-ditions. Though the biocontrol effect needs to be eva-luated for a longer growth period and under field con-ditions, this study provided an effective and alternativeapproach for the suppression of banana wilt.

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