Biological Control of Meloidogyne incognita by Aspergillus niger F22 Producing Oxalic Acid
RESEARCH ARTICLE
Biological Control of Meloidogyne incognita
by Aspergillus niger F22Producing Oxalic
Acid
Ja Yeong Jang 1,2,Yong Ho Choi 3,Teak Soo Shin 4,Tae Hoon Kim 4,Kee-Sun Shin 5,Hae
Woong Park 6,Young Ho Kim 7,Hun Kim 3,Gyung Ja Choi 3,Kyoung Soo Jang 3,
Byeongjin Cha 2,In Seon Kim 1,Eul Jae Myung 4,Jin-Cheol Kim 1*
1Department of Agricultural Chemistry,Institute of Environmentally Friendly Agriculture,College of
Agriculture and Life Sciences,Chonnam National University,Gwangju,Republic of Korea,2Department of
Plant Medicine,Chungbuk National University,Cheongju,Republic of Korea,3Center for Eco-friendly New
Materials,Korea Research Institute of Chemical Technology,Daejeon,Republic of Korea,4Crop Protection
Research Team,Dongbu Advanced Research Institute,Dongbu Farm Hannong Company,Ltd.,Nonsan-si,
Republic of Korea,5Biological Resources Center,Korea Research Institute of Bioscience and
Biotechnology,Daejeon,Republic of Korea,6World Institute of Kimchi,an Annex of Korea Food Research
Institute,Gwangju,Republic of Korea,7Department of Agricultural Biotechnology and Research Institute of
Agriculture and Life Sciences,Seoul National University,Seoul,Republic of Korea
*kjinc@jnu.ac.kr
Abstract Restricted usage of chemical nematicides has led to development of environmentally safe alternatives.A culture filtrate of Aspergillus niger F22was highly active against Meloido-gyne incognita with marked mortality of second-stage juveniles (J2s)and inhibition of egg hatching.The nematicidal component was identified as oxalic acid by organic acid analysis and gas chromatography-mass spectroscopy (GC-MS).Exposure to 2mmol/L oxalic acid resulted in 100%juvenile mortality at 1day after treatment and suppressed egg hatching by 95.6%at 7days after treatment.Oxalic acid showed similar nematicidal activity against M .hapla ,but was not highly toxic to Bursaphelenchus xylophilus .The fungus was incubated on solid medium and dried culture was used for preparation of a wettable powder-type (WP)formulation as an active ingredient.Two WP formulations,F22-WP10(ai 10%)and oxalic acid-WP8(ai 8%),were prepared using F22solid culture and oxalic acid.In a field naturally infested with M .incognita ,application of a mixture of F22-WP10+oxalic acid-WP8at 1,000-and 500-fold dilutions significantly reduced gall formation on the roots of watermelon plants by 58.8and 70.7%,respectively,compared to the non-treated control.The disease control efficacy of the mixture of F22-WP10+oxalic acid-WP8was significantly higher than that of a chemical nematicide,Sunchungtan (ai 30%fosthiazate).These results suggest that A .niger F22can be used as a microbial nematicide for the control of root-knot nema-tode
disease.
OPEN ACCESS
Citation:Jang JY,Choi YH,Shin TS,Kim TH,Shin
K-S,Park HW,et al.(2016)Biological Control of
Meloidogyne incognita by Aspergillus niger F22
Producing Oxalic Acid.PLoS ONE 11(6):e0156230.
doi:10.1371/journal.pone.0156230
Editor:Kap-Hoon Han,Woosuk University,
REPUBLIC OF KOREA
Received:January 21,2016
Accepted:May 11,2016
Published:June 3,2016
Copyright:?2016Jang et al.This is an open
access article distributed under the terms of the
Creative Commons Attribution License ,which permits
unrestricted use,distribution,and reproduction in any
medium,provided the original author and source are
credited.
Data Availability Statement:All relevant data are
within the paper and its Supporting Information files.
Funding:This study was performed with support
from the Cooperative Research Program for
Agricultural Science and Technology Development
(Project PJ01020702),Rural Development
Administration,Republic of Korea (http://www.rda.go.
kr ).The funder provided support in the form of
salaries for authors JYJ,YHC,HK,GJC,KSJ,and
JCK.Dongbu Farm Hannong Company Ltd provided
support in the form of salaries for authors TSS,THK
and EJM,but did not have any additional role in the
Introduction Plant disease caused by nematode infection is a major problem in crop production.Plant-para-sitic nematodes caused annual yield loss estimated at 8.8–14.6%of total crop production [1].Root-knot nematodes (RKNs;Meloidogyne spp.),scientifically and economically the most important phytonematode [2],occur globally,especially in tropical and sub-tropical agricul-tural areas,and cause significant yield losses annually [3].Southern root-knot nematode (M .incognita ),one of the most important species of RKNs,infects the roots of almost all cultivated
plants.RKNs impede uptake of water and nutrients due to formation of giant cells in the roots
of many crops,and facilitate infection by pathogenic microorganisms.
Control of RKNs is difficult because they have short generation times and high reproduction
rates [4,5].Several management strategies can be applied to control RKNs;chemical nemati-
cides have generally been used.However,these agents are increasingly being withdrawn or
restricted due to their potential negative impact on human health and the environment [6].In
addition,policies designed to support sustainable or environmentally friendly farming prac-
tices in many countries and increased demand for eco-friendly measures has necessitated
development of safe and effective alternatives [7].Thus,much research has aimed to identify
antagonistic microorganisms and their metabolites with nematicidal activity against RKNs [8–
10].However,this achieved limited success when applied in the field,and consequently few
commercial products have been developed and used in agricultural practice [11].Therefore,
identification of antagonists for control of plant parasitic nematodes and their commercializa-
tion are necessary.
Microbial agents generally have slower nematicidal activity than chemical nematicides.In
certain agricultural applications,slower nematicidal activities have prevented adoption of bio-
pesticides.In addition,the inconsistent performance of microbial agent must be overcome if
these are to be used as commercial nematicides [12].Development of appropriate formulations
would facilitate large-scale utilization of biocontrol agents.A suitable formulation benefits the
application and handling of the bio-agent,and increases its efficacy by protecting the active
ingredient from adverse environmental factors [13,14].Although developing appropriate com-
mercial formulations of antagonistic microorganisms is important,a few studies have been
reported [15,16].Recently,Bacillus subtilis strain CPA-8formulations were prepared by spray
drying using various combinations of skimmed milk and MgSO 4as carriers/protectants.The
CPA-8formulation (1.6–3.3×109CFU/g)exhibited efficacy similar to fresh cells in terms of
controlling brown rot disease [15].In addition,a wettable powder-type formulation of Pseudo-
monas fluorescens EPS62e using lactose as a lyoprotectant showed improved biocontrol efficacy
and survival at low relative humidities [16].
In the course of screening of nematicidal fungi,we found that Aspergillus sp.F22culture fil-
trate had marked nematicidal activity against M .incognita .Therefore,the aims of this study
were 1)to investigate the nematicidal potential of strain F22,2)to characterize the nematicidal
metabolites produced by the fungus,and 3)to evaluate the efficacy of the strain F22formula-
tion as a biological control agent for M .incognita under field conditions.
Materials and Methods
Antagonistic fungal strain
Aspergillus sp.F22was isolated from soil on Gyejok Mountain,Daejeon,Korea.Strain F22was
deposited in the Korean Collection for Type Cultures (KCTC)as KCTC12771BP.A stock cul-
ture of the strain was stored in 8%dimethyl sulfoxide suspension at -80°C.Strain F22was
maintained on potato dextrose agar (PDA)slants and stored at 4°C.The strain was grown
at
study design,data collection and analysis,decision to
publish,or preparation of the manuscript.
Competing Interests:The authors received funding
from Dongbu Farm Hannong Company Ltd for this
study.There are no patents,products in development
or marketed products to declare.This does not alter
the authors'adherence to all the PLOS ONE policies
on sharing data and materials.
25°C on PDA medium,and then inoculated into500mL Erlenmeyer flasks containing200mL of potato dextrose broth(PDB).Strain F22was grown at25°C in PDB for7days with shaking at150rpm and filtered through sterile gauze to yield culture filtrate.
Identification of the antagonistic fungal strain
Cells were disrupted by bead beating and DNA extraction was carried out as described by Yu and Mohn[17].PCR-mediated amplification of the5.8S-ITS region was carried out using the procedures described by Scorzetti et al.[18].PCR products were purified using a QIA quick PCR purification kit(Qiagen,Hilden,Germany)and sequenced on an Applied Biosystems310 DNA sequencer using a Taq DyeDeoxy Terminator cycle sequencing kit(Applied Biosystems, Foster City,CA,USA),according to the manufacturer’s protocol.Existing sequences for refer-ence species were retrieved from GenBank.Sequences were aligned using the multiple sequence alignment software CLUSTAL X and manually corrected[19].A phylogenetic tree was obtained from the DNA sequences using the neighbor-joining method[20]with Kimura’s two-parameter model for calculating genetic distances[21]and1,000bootstrap replications using the PHYLIP3.57c software[22].The tree was visualized using the TreeView software [23].Talaromyces flavus NRRL2098(EU021596)was used as the designated outgroup. Nematodes
M.incognita,which was isolated and identified by Hwang et al.[24],was maintained on tomato(Lycopersicon esculentum Mill.cv.Seokwang)in a greenhouse(28±5°C).Eggs were extracted from tomato roots infected with M.incognita using1%sodium hypochlorite solu-tion.The nematode eggs were collected by passage through a45μm sieve,followed by a25μm sieve.Collected eggs were rinsed with distilled water and used for in vitro experiments and allowed to hatch using modified Baermann funnels[25]at28°C within5days to obtain sec-ond-stage juveniles(J2s).
Meloidogyne hapla was isolated from root galls of ginseng cultivated in Jinan-gun,Jeon-buk, Korea[26].Four-week-old tomato plants(Solanum lycopersicum cv.Rutgers)grown in a growth chamber were inoculated with J2s of M.hapla and cultivated at25±2°C in a green-house.J2s of M.hapla were obtained using the same methods applied to M.incognita.B.xylo-philus was isolated from chips of infested pine trees and cultured on PDA plates containing Botrytis cinerea at25°C.B.cinerea were obtained from the Southern Forest Research Center of the Korea Forest Research Institute.Nematodes were extracted using the Baermann funnel method,followed by sieving(38μm).
Effect of F22culture filtrate on M.incognita J2s and eggs
Effects of Aspergillus sp.F22culture filtrate(1.25–20%)on M.incognita J2s and eggs were eval-uated.Approximately50J2s and150eggs per well were used for the bioassay using96-well tis-sue culture plates(Becton Dickinson,Franklin Lakes,NJ).Sterile distilled water(SDW)was used as the negative control.The plates were gently shaken and incubated at100%humidity in a plastic box at room temperature in the dark.All experiments were conducted with three repli-cates and the experiment was twice repeated.J2s were counted after1day of incubation and judged as dead if their body was straight with no movement despite physical stimulation with a fine needle.Mortality rates(M)were corrected using Abbott’s formula[27]:
M?
eMtàMcT
e100àMcT
?
100
Where Mt means mortality percentage in treatment and Mc means mortality percentage in
control.The rate of inhibition of nematode egg hatching was assessed under a light microscope
at 7days after treatment.Hatch inhibition (HI)was calculated according to the formula:HI ?eC ‐T TC
?100Where C and T are the percentages of eggs hatched in the control and treatment,respectively.
Egg hatch rate (EH)was calculated as follows:EH ?J eE tJ T
?100Where J and E mean the juveniles and eggs of M .incognita ,respectively.
Organic acid analysis
Aspergillus sp .F22culture filtrate was passed through a membrane filter (0.45μm).The filtrate
was diluted fivefold and organic acids were analyzed by high-performance liquid chromatogra-
phy (HPLC)(Agilent 1100series HPLC,Santa Clara,CA,USA).An Aminex HPX-87C column
(4.6×250mm,Bio-Rad,Marnes-la-Coquette,France)was used for analysis.Elution was car-
ried out isocratically using 5mmol/L sulfuric acid.The flow rate and column temperature were
0.6mL/min and 28°C,respectively.Detection was performed by a UV detector at 210nm
(G1314A,Agilent HPLC 1100series).Quantitative analysis of organic acids was performed
using standard curves.
To confirm the presence of oxalic acid,the filtrate was further analyzed by GC-MS after
methyl derivatization.An aliquot (0.5mL)of culture filtrate was mixed with 0.5mL of 29
mmol/L malonic acid,and then added to 2mL of methanol and 0.4mL of 50%sulfuric acid
(H 2SO 4).The sample was heated at 60°C for 30min with caps closed and then 1mL of distilled
water and 0.5mL of chloroform added,followed by agitation for 30s.The chloroform layer
was taken and 1μL of the sample was injected into the GC-MS.GC-MS analysis was conducted
using an Agilent Technologies system (Wilmington,DE,USA)consisting of a model 7890gas
chromatograph and a model 5975mass selective detector.The column was a DB-WAX (id
0.25mm ×length 30m,film thickness 0.25μm).GC temperature program was as follows:ini-
tial temperature of 40°C,which was held for 1min,increased to 150°C at a rate of 2°C/min,
then to 200°C at a rate of 4°C/min,and finally to 250°C at a rate of 6°C/min,and held for 10
min.The split ratio was 1:12,injection temperature was 200°C,transfer line temperature was
250°C,and ion source temperature was 200°C.The mass spectrometer was operated at 70eV in
the electron impact mode.The spectrum obtained from the sample was identified by compari-
son with mass spectra in the NIST library and a standard solution (0.1mol/L oxalic acid).
Effects of oxalic acid,citric acid and tartaric acid on M .incognita J2s and
eggs
Stock solutions of oxalic acid (!99%purity;Sigma-Aldrich,St.Louis,MO),citric acid (99%
purity;Sigma-Aldrich),and tartaric acid (99%purity;Sigma-Aldrich)were prepared using dis-
tilled water.A stock solution of trans -cinnamaldehyde (cinnamaldehyde;99%purity;Sigma-
Aldrich),which is used as a natural nematicide [28],was prepared using ethanol.Stock solu-
tions were tested at a concentration range of 0.08–50mmol/L.The final concentration of etha-
nol did not exceed 1%of the volume.Ethanol (1%)was used as a negative control.Ninety-or
ninety-nine-microliter aliquots of suspensions of J2s (~50per well)or eggs (~150per well)
were placed in each well,followed by addition of 10μL of stock solutions of organic acids
or
1μL of stock solution of cinnamaldehyde.The juvenile mortality and egg hatching rates were evaluated as described above.The experiment was carried out with five replicates and the experiment was twice repeated.M.incognita juvenile mortality and egg hatching rates were determined after1and7days of incubation,respectively.
Effect of oxalic acid on J2s of M.hapla and B.xylophilus
The nematicidal activity of oxalic acid(0.4–10mmol/L)was investigated against M.hapla and B.xylophilus as described above for M.incognita.Distilled water(10%)was used as the negative control.Ninety-microliter aliquots of juvenile suspension(~50per well)were placed in each well,followed by addition of10μL of stock solution.The experiment was carried out with three replicates and the experiment was twice repeated.The mortality rate of juvenile nema-todes was evaluated after1day of incubation.
Solid-state fermentation of Aspergillus sp.F22and formulation
The fungal strain F22was cultured on PDA at25°C for7days and then spores were harvested using distilled water(1.0×106spores/mL).Sterile wheat-rice bran medium(wheat bran100g, rice bran100g,and distilled water100mL in a1L Erlenmeyer flask)was inoculated with2mL of spore suspension,followed by incubation at30°C for7days in the dark to yield4.5×109 spores/g.Solid-state culture of Aspergillus sp.F22(100g,10%)was mixed with720g of kaoline, 90g of sodium dodecyl sulfate(CR-SDS;Yoosung Chemical R&T Co.,Ltd,Daejeon,Korea)as a wetting agent,and90g of sodium poly(naphthalene formaldehyde)sulfonate(CR-100;Yoo-sung Chemical R&T Co.,Ltd)as a dispersal agent.The wettable powder-type formulation of Aspergillus sp.F22(F22-WP10)was milled in a blender(IKA1-Werke GmbH&Co.KG,Stau-fen,Germary).The A.niger F22formulation had viable spore count of4.1×108CFU/g.
Oxalic acid(95%purity)was purchased from Chunbo Fine Chemical(Eumseong-gun, Korea).A wettable powder-type formulation of oxalic acid(OA-WP8)was prepared by mixing 80g of oxalic acid,270g of synthetic hydrated silicon dioxide(white carbon;Rhodia Asia Pacific Pte Ltd,The Concourse,Singapore),90g of CR-SDS,90g of CR-100,and570g of kao-line[29].
Efficacy of F22for biocontrol of M.incognita on watermelon plants under field conditions
The experiment was conducted in a field naturally infected with RKNs(Meloidogyne spp.), located in Chaeun-myeon,Nonsan-si,Chungnam,Korea in June2015.The owner of the land gave permission to conduct the study on this site.The field was severely affected by RKNs dur-ing previous seasons.Watermelon seeds(cv.Uriggul)were sown into seed trays containing clay-loam soil in a greenhouse and after3weeks transplanted into the plots.To calculate the initial nematode density in the soil,five soil cores(250cm3each)were collected from each plot before transplantation of watermelon.The nematode densities in soil were assessed by soil core sampling(7.2×10cm)at a depth range of10–20cm per treatment plot selected randomly. Nematodes in soil were extracted from a200g soil subsample by sieving and centrifugation [30]and4×3mL aliquots of a50mL suspension counted on a counting dish under a micro-scope(Olympus CX31).The initial nematode densities were235±16J2s and55±3eggs per 100mL soil.The field experiment was performed using a randomized complete block design with four replicates;each treatment consisted of10m2(2×5m2)plots.The treatments were: (1)untreated control,(2)1,000-and(3)500-fold dilutions of F22-WP10,(4)500-fold dilution of OA-WP8,(5)1,000-and(6)500-fold dilutions of a mixture of F22-WP10and OA-WP8, and(7)2,000-fold dilution of Sunchungtan(EC:ai30%fosthiazate,70%surfactant;Dongbu
Farm Hannong Co.,Korea).An aliquot(200mL)of each treatment was applied to the soil around watermelon roots following the start of gall formation four times at10-day intervals. The negative control was200mL of water only.At60days after the first treatment,the galling index(GI)was assessed according to a0–5galling scale:0=0–10%galled roots,1=11–20%, 2=21–50%,3=51–80%,4=81–90%,and5=91–100%[31].The control value was calculated according to the formula:control value(%)=100×(galling index of control—galling index of treatment)/galling index of control.
Statistical Analysis
Data were subjected to one-way ANOVA and the means of the treatments were separated by Duncan’s multiple range test(p<0.05)using the SPSS software(SPSS,version21.0for Win-dows,Chicago,IL).The50%effective concentrations(EC50)were determined by probit analy-sis(95%confidence limits)using the SPSS software.
Results
In vitro nematicidal activity and identification of F22
The culture filtrate of strain F22at various concentrations showed nematicidal activity against J2s and inhibited egg hatching.Its effects on killing J2s and suppressing egg hatching increased in a dose-dependent manner.The J2mortality rate was25.3%at1.25%culture filtrate and
>90%at2.5–20%culture filtrate at1day after treatment(Fig1A).Complete inhibition of egg hatching was observed at5–20%culture filtrate after7days of exposure as compared to the control(Fig1B).The rate of inhibition of egg hatching was reduced to77.5%and14.0%at 2.5%and1.25%culture filtrate,respectively.Fungal strain F22exhibited98.7%similarity to Aspergillus niger CBS554.65T(AJ223852)according to5.8S-ITS rRNA gene sequence analysis (S1Fig).The F22sequence was deposited in GenBank under accession number KU221232. Identification of the nematicidal metabolite
To identify the nematicidal metabolite produced by strain F22,organic acid analysis was con-ducted because the culture filtrate showed strong acidity(pH1.7).Oxalic aicd(7.9g/L)was the major organic acid in culture filtrate of strain F22(Table1).Citric acid and tartaric acid were also detected,albeit at lower concentrations(461.6and388.1mg/L,respectively)than that of oxalic acid.Based on its content in culture filtrate and its nematicidal activity,OA was identi-fied as a major nematicidal component.GC-MS analysis after methyl derivatization confirmed the presence of oxalic acid in the F22culture filtrate.The EI-mass spectrum of oxalic acid methyl ester showed a molecular ion peak at m/z118[M+]and a base peak at m/z59
[M-COOCH3+](S2Fig),which was identical to that of the standard compound.
Effects of oxalic acid,citric acid,and tartaric acid on J2s and eggs of M. incognita
Exposure to2mmol/L oxalic acid solution caused100%mortality of M.incognita J2s at1day after treatment(Fig2A).The nematicidal activity of oxalic acid against M.incognita J2s was lower than that of cinnamaldehyde,a natural nematicide.Exposure of M.incognita J2s to50 mmol/L citric acid and tartaric acid resulted in96.0%mortality,whereas application of10 mmol/L resulted in mortality rates of59.5%and86.9%,respectively.The EC50values of oxalic acid and cinnamaldehyde were0.87and0.17mmol/L,respectively.The EC50values of citric acid and tartaric acid were14.35and4.32mmol/L,respectively,at1day after treatment.
In addition,treatment with 2mmol/L oxalic acid resulted in 95.6%inhibition of egg hatch-
ing.Moreover,10and 50mmol/L oxalic acid resulted in 100%inhibition of egg hatching at 7
days after treatment (Fig 2B ).In comparison,0.4mmol/L cinnamaldehyde completely inhib-
ited egg hatching.Treatment with 50and 10mmol/L citric acid and tartaric acid resulted in
89.3%and 100%,62.4%and 87.2%,inhibition of egg hatching,respectively.The EC 50values of
oxalic acid,citric acid,tartaric acid,and cinnamaldehyde were 1.15,17.08,4.20,and 0.16
mmol/L,respectively,at 7days.Microscopic observation indicated that OA oxalic acid
completely destroyed internal organs,resulting in production of multiple vacuoles in the nema-
tode body,which was straightened and stiffened (Fig 3).In comparison,the exposure
to
Fig 1.Effects of F22culture filtrate on J2mortality (A)and egg hatching (B)of M .incognita .Values are
means ±standard deviation of three replicates.Means with the same letter are not significantly different
(p <0.05)according to Duncan ’s multiple range test.
doi:10.1371/journal.pone.0156230.g001
cinnamaldehyde at the same concentration caused relatively weak disruption of internal
organs.The negative control (1%ethanol)did not induce such symptoms in M .incognita J2s.
In addition,the nematicidal activity of oxalic acid against J2s of M .hapla and B .xylophilus
was investigated.As shown in Fig 4,M .hapla was highly sensitive to oxalic acid,to a level simi-
lar to M .incognita ,but B .xylophilus was relatively resistant compared to the two Meloidogyne
species.The EC 50values of oxalic acid were 1.15mmol/L for M .hapla and 9.37mmol/L for B .
xylophilus .
Biocontrol of root knot nematode disease by F22under field conditions
Under field conditions,F22-WP10and OA-WP8,at a 500-fold dilution,resulted in moderate
inhibition of gall formation on the roots of watermelon plants (Fig 5).Co-treatment with the
two formulations resulted in significantly higher inhibitory activity than those of either formu-
lation alone.Application of a 1:1mixture of the two formulations,at 1,000-and 500-fold dilu-
tions,significantly (p <0.05)reduced gall formation by 58.8%and 70.7%,respectively.This
activity was higher than that of Sunchungtan (45.1%),a chemical nematicide.
Discussion
Sustainable pest management in agriculture is hampered by nematodes,especially in intensive
greenhouse cultivation [32].Prohibition of the use of common chemical nematicides and their
decreased efficacy due to continuous utilization are barriers to control of RKNs [33].Moreover,
global warming has conditions favorable for RKNs.Identification of environmentally friendly
and non-toxic control measures of RKNs is thus required.Our results indicated that exposure
to A .niger F22culture filtrate reduced the rates of J2viability and egg hatching.This effect was
attributed to the production of oxalic acid as a nematicidal metabolite.Mankau (1969)reported
oxalic acid to be a nematicidal metabolite of A .niger and its nematicidal effect both in vitro and
in soil against Aphelenchus avenae [34].Kurt et al.[35]reported that A .niger cause moderate
toxicity against Aphelenchus avenae and A .besseyi by producing oxalic acid and citric acid.
Zuckerman et al.[36]reported that both citric acid and oxalic acid were nematicidal metabo-
lites in culture filtrate from an isolate of A .niger (designated PD-42),but their nematicidal
effects against Caenorhabditis elegans were weak;>10g/L oxalic acid resulted in 100%mortal-
ity of C .elegans juveniles,but 16g/L citric acid did not show 100%mortality.However,the
combination of oxalic acid (4g/L)and citric acid (4g/L)was nematicidal,suggesting a syner-
gistic effect.Furthermore,Mokbel et al.[37]reported that culture filtrate of A .niger and oxalic
acid and citric acid have nematicidal activity against M .arenaria as a similar result.In
this
Table 1.Production of organic acids by A .niger F22in potato dextrose broth medium.
Organic acid
Production (mg/L)a ND:not detected.
doi:10.1371/journal.pone.0156230.t001
study,we report that oxalic acid produced by A .niger F22has strong nematicidal activity
against M .incognita and M .hapla .
The nematicidal activity of low-molecular-weight organic acids has been reported [38–40].
Browing et al.[38]reported that exposure to 0.1and 1mol/L butyric acid resulted in 100%
mortality of M .hapla and M .incognita .Seo and Kim [39]reported that a mixture of acetic acid
and lactic acid had greater toxicity on M .incognita J2s than either agent alone.Also,Bansal
and Bajaj [40]reported that six volatile fatty acids reduced the rate of egg hatching during 12
days of incubation in the following order:propionic acid >acetic acid >caprylic acid
>
Fig 2.Effects of oxalic acid,citric acid,tartaric acid and cinnamaldehyde on M .incognita J2mortality and egg hatching
rates.Values are means ±SD of five replicates.Means with the same letter are not significantly different (p <0.05)according to
Duncan ’s multiple range test.
doi:10.1371/journal.pone.0156230.g002
isobutyric acid >valeric acid >butyric acid.To our knowledge,this is the first report of nema-
ticidal activity and egg hatching inhibitory activity of oxalic acid against M .incognita .
Although the mode of action of oxalic acid is unknown,the strong acidity of oxalic acid
(pKa =1.25)may cause rapid destruction of cells and tissues of the nematode bodies and eggs
[39].This may be caused by osmoregulation disruption and then fluid accumulation.Our
microscopic analysis supported these hypotheses (Fig 3).We investigated pH values of three
organic acids and cinnamaldehyde.Except cinnamaldehyde,as the concentration of the three
organic acids in solutions increased,the more acidic the solutions became (S3Fig ).At the same
concentrations,pH values of oxalic acid were highest,followed in order by tartaric acid and cit-
ric acid.This trend coincided with the extents of mortalities of three organic acids;the mortal-
ity of oxalic acid was highest among the three organic acids,followed in order by tartaric acid
and citric acid (Fig 2).These results strongly suggest that the nematicidal activity of oxalic acid
is closely related to its strong acidity.However,the other mechanism should not be ruled out
completely.A .niger F22in liquid medium produces oxalic acid,citric acid and tartaric acid.
Compared to oxalic acid,citric acid and tartaric acid were not highly toxic to M .incognita .
Thus oxalic acid was a major nematicidal component of F22culture filtrate.Oxalic acid is pres-
ent in various foods and plants (such as amaranth,cassava,chives,and parsley)at levels of
>1.0g/100g [41].Oxalic acid is used rarely in Canada,Europe,and the USA as a
biopesticide
Fig 3.Morphological variations in M .incognita J2s treated with 2mmol/L oxalic acid (A),2mmol/L cinnamaldehyde (B),and water
(C).Bar =50μm.
doi:10.1371/journal.pone.0156230.g003
for the control of varroa mite on honey bees [42–43].Therefore,A .niger F22culture filtrate
and oxalic acid can be used as biopesticides for the control of RKNs.
In the field experiment,treatment of soil with the F22-WP10+OA-WP8mixture resulted
in greater efficacy than either agent alone.The treatment of F22-WP +OA-WP8mixture not
only may rapidly reduce the M .incognita J2population density,but may also maintain a low
nematode population through production of oxalic acid by A .niger F22.On the other hand,
Gadd [44]reported that oxalic acid and citric acid produced by fungi may bind to metal ions
including Zn ++,and it suppressed egg hatching due to interaction with Zn ++ions [45].How-
ever,the underlying mechanism is unknown.Also,oxalic acid would likely induce systemic
resistance in plants against diseases caused by fungi,bacteria,and viruses and may enhance
host defense by increasing defense-related enzyme activities and production of secondary
metabolites,such as phenolics [46–49].This may hinder nematode invasion and subsequent
infection.Zuckerman et al.[36]reported that incorporation of A .niger PD-42in seed coats
suppressed gall formation on pepper by M .incognita and increased yield.These previous
reports as well as our results suggest the possibility of commercial use of the mixture of A .niger
F22solid culture and oxalic acid in farming.This is the first study of a commercial A .niger and
oxalic acid formulation for control of M .incognita .
Because of the issue of pesticide residue,Sunchungtan (ai.fosthiazate 30%)is generally used
once before plantation,and it cannot be used during crop cultivation.Although the pesticide
was treated four times at 10-day intervals in this study,its disease control efficacy against
RKN
Fig 4.Oxalic acid -induced mortality of M .hapla and B .xylophilus J2s.Values are means ±SD of three
replicates.Means with the same letter are not significantly different (p <0.05)according to Duncan ’s multiple range
test.
doi:10.1371/journal.pone.0156230.g004
disease in watermelon was significantly lower than that of a 500-fold dilution of a 1:1mixture
of F22-WP10+OA-WP8.This suggests that the F22-WP10+OA-WP8mixture would show
high disease control efficacy in the field.In addition,the mixture of F22-WP10+OA-WP8can
be used during crop cultivation.This will facilitate effective control of RKNs in the field.Even
though A .niger has been generally regarded as safe,it have the potential for mycotoxin produc-
tion such as fumonisins and ochratoxins [50].Therefore,we need to scrutinize its production
potential before commercialization.
Biopesticides comprise a small proportion of the pesticides used for crop protection.Biolog-
ical products are highly target-specific and their use is desirable,but acceptable formulations
are difficult to develop [51].The formulated product must be stored under appropriate condi-
tions prior to application for maximum efficacy.Our results suggest the mixture of F22-WP10
and OA-WP8to be a promising nematicide for the control of RKNs in conventional farming
systems.Further studies are required 1)to examine the toxicity of the two formulations,2)to
evaluate the disease control efficacy of the mixture of the two formulations in various fields,3)
to examine the disease control spectrum of the two formulations against various nematode dis-
eases,and 4)to develop more effective and eco-friendly
formulations.
Fig 5.Efficacy of agents individually or in combination on watermelon plants (cv.Uriggule)in a field naturally infected condition by M .incognita .F (×1,000),1,000-fold dilution of F22-WP10;F (×500),500-fold dilution;FO,F22-WP10+OA-WP8;O,OA-WP8;and S (×2,000),2,000fold-dilution of Sunchungtan (150μg/mL fosthiazate).F22-WP10:a wettable powder-type formulation of A .niger F22(ai 10%),OA-WP8:a wettable powder-type formulation of oxalic acid (ai 8%).Data are means ±SD of four replicates.Relationships among
values were subjected to one-way ANOVA and Duncan ’s multiple range test.Means with the same letter did not differ significantly (p <0.05).
doi:10.1371/journal.pone.0156230.g005
Supporting Information
S1Fig.Neighbour-joining phylogenetic tree based on5.8S-ITS sequences of F22and its closest relatives.Numbers at nodes represent the proportions of1,000bootstrap samples in which a clade was found.Bar,0.01substitutions per nucleotide position.Talaromyces flavus NRRL2098was used as the outgroup.
(TIF)
S2Fig.GC-EI-MS total ion chromatogram of Aspergillus niger F22culture filtrate(A)and mass spectrum of the peak at the retention time25.347(B).Standard solution was prepared at a concentration of0.1mol/L oxalic acid.
(TIF)
S3Fig.pH values of oxalic acid,citric acid,tartaric acid,and cinnamaldehyde with differ-ent concentrations.Values are means±standard deviation of three replicates.
(TIF)
Author Contributions
Conceived and designed the experiments:JYJ JCK.Performed the experiments:JYJ YHC TSS THK KSS HWP YHK HK GJC KSJ BC ISK EJM JCK.Analyzed the data:JYJ KSS TSS GJC JCK.Contributed reagents/materials/analysis tools:KSS HWP HK GJC EJM JCK.Wrote the paper:JYJ KSS JCK.
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