The Synergism of Chemical Herbicides and Aureobasidium pullulans for Control Cleavers (Galium aparine L.)in Wheat

Liang CHENG

1.Key Laboratory of Agricultural Integrated Pest Management in Qinghai Province,Institute of plant protection,Qinghai Academy of Agriculture and Forestry Sciences,Xining 810016,China;

2.Scientific Observing and Experimental Station of Crop Pest in Xining,Ministry of Agriculture,Xining 810016,China

The Synergism of Chemical Herbicides and Aureobasidium pullulans for Control Cleavers (Galium aparine L.)in Wheat

Liang CHENG1,2

1.Key Laboratory of Agricultural Integrated Pest Management in Qinghai Province,Institute of plant protection,Qinghai Academy of Agriculture and Forestry Sciences,Xining 810016,China;

2.Scientific Observing and Experimental Station of Crop Pest in Xining,Ministry of Agriculture,Xining 810016,China

Aureobasidium pullulans,a biocontrol agent for the annual weedGalium aparineL.was evaluated in vitro for its compatibility with commercial formulation of five herbicides at 1X(recommended field rate),0.5X,0.2X,0.1X,0.067X,and 0.05X concentrations.Germination ofA.pullulanswith paraquat,2,4-D,quizalofop-p,and clethodim treatment appeared reduced compared with germination ofA.pullulanswith fluroxypyr treatment at all concentrations.Stunted and shorter germ tubes in comparison with the control were observed with 2,4-D,quizalofop-p,and clethodim at 0.2X.All concentration of paraquat,2,4-D,quizalofop-p,and clethodim except 0.05X,significantly decreased radial growth ofA.pullulanscompared with its growth on the untreated PDA medium.Field trials to further developA.pullulansas biocontrol agent for controlG.aparineL.was conducted to test the effectiveness of this fungus in wheat plots for 2 years at the same location in Xining.Treatments included spore suspensions ofA.pullulansalone,a mixture of both fungus and fluroxypyr in wheat.Biocontrol agent effectiveness was estimated at approximately 7 and 14 days after treatment,as disease incidence,percent weed control,and weed biomass reduction.Significant reduction in weed biomass occurred in combination treatments,and potential exists to tank mixA.pullulanswith fluroxypyr.Leaf surface moisture and air temperatures following application may account for inconsistencies in field results between years.This fungal organisms show potential as bioherbicides for weeds inG.aparineL.

Aureobasidium pullulans;Galium aparineL.,tenerum(Gren.et Godr.) Rchb.;Chemical herbicide;Synergism;Integrated weed management

C leavers(Galium aparineL.)is a frequent and harmful weed species in wheat and rape fields,especially widely distributed throughout most of the major wheat production regions in China.Cleavers sticks to the above-ground parts of cultivated plants and suffocates them, thus making the harvest difficult by clogging machinery and contaminating the material harvested,which may cause grain yield losses of 19.44%-41.67%[1].The problems with cleavers have been accentuated because of weed biotype resistant to tribenuronmethyl herbicide[2-3].There is a need for alternative methods for controlling cleavers resistant to tribenuron-methyl or the ALS-inhibitor in wheat and rape crops.

The biocontrol agents approach has shown promise for control of herbicide-resistant weeds in production agriculture and is an attractive and appropriate choice for growers because biocontrol agents can be applied with conventional herbicide application equipment and used in a manner similar to postemergence herbicides[4].In addition,most biocontrol agents introduced using either classical or inundative biocontrol methods have an excellent safety record regarding nontarget damage[5].

Often a weed species is host to a number of natural enemies and this natural association of interactive agents can be exploited to achieve integrated weed control.Rather as a replacement for chemical herbicides, biological control of weeds is gaining increasing importance as a viable part of well-designed,integrated weed management systems.Fungi figure prominently among potential biocontrol agents of major agricultural weeds.Integration of selected biocontrol measures into general integrated weed management strategies requires that biocontrol agents are compatible with herbicides used in the target crop. Factors that increase the germination and virulence of the biocontrol agents may also increase efficacy[6]. One method to improve pathogen efficacy is the use of selected chemical herbicides.These chemical herbicides include 2,4-D,glyphosate,and halosulfuron-methyl that may improve bioherbicidal efficacy through various mechanisms.Chemical herbicide:bioherbicide interaction studies have primarily focused on their influence on spore germination,appressorial formation and mycelia growth[7-8].

The effect of natural and synthetic chemical herbicides on biocontrol agent is an important element in the development of integrated weed management strategies[9].A major concern is whether a biocontrol agent can be integrated within the normal herbicide spraying schedule used for cropprotection.Compatibility of herbicides with fungal biological control agents has been examined to some extent[10]. Some herbicides and growth regulators with specific chemistries and molecular modes of action have been shown to act synergistically with biocontrol agents to enhance weed control[11].Sharon et al.[12]demonstrated that the suppression of biosynthesis of a phytoalexin in Senna(=Cassia)obtusifolia(L.)H.S.Irwin&Barneby by the herbicide glyphosate at sublethal rates led to subsequent increase in susceptibility of this weed to the mycoherbicide Alternaria cassiae Jurair&Khan. Another example of synergy between bioherbicide and chemicals is in the application of Fusarium avenaceum in combination with the herbicide quizalofop-p-ethyl to achieve 83% control of Avena fatua L.,compared to only 66%control with the F.avenaceum alone[13].

Singh et al.[14]found a synergistic interaction between Microsphaeropsis amaranthi and glyphosate that enhanced disease severity(DS)and weed control in soybean.Greater than 90%weed control was seen in treatments where both M.amaranthi and glyphosate were used.Although glyphosate provided good weed suppression,weed escapes were common;however,combination applications of glyphosate with M.amaranthi provided excellent control.This demonstrated the strong potential of the bioherbicide to be integrated into soybean production,particularly where control by glyphosate is inadequate. In addition,certain herbicides can be toxic to certain fungi.Smith and Hallett[15]and Wyss et al.[16]found that glyphosate reduced spore germination of M.amaranthi and P.amaranthicola, respectively.Therefore,herbicides used with a fungus must be tested for toxicity to that particular strain both singly and in combination with any other measures which is to be included in the development of integrated weed management strategies.

Aureobasidium pullulans has been used for control of Galium aparine L. in controlled environment and greenhouse trials[17].However,no information is available on control of Galium aparine L.in wheat by biological agent alone or combined with use of biocontrol agent and herbicides.The primary purpose of this investigation was to determine whether herbicides used at different doses affected conidial germination of A.pullulans,and to develop the types of initial estimates of compatibility that in vitro studies will provide.Selected herbicides were also tested at different doses for their effects on vegetative growth and field efficacy of A.pullulans.

Materials and Methods

Fungal culture

A single-spore isolate of Aureobasidium pullulans(isolate PA-2) used in this study was isolated a naturally diseased poplar plant found in Ping’an,Qinghai Province,China. Stock cultures were maintained for long-term storage in sterilized sand soil in sterile glass tubes in the dark at 4℃.For inoculum preparation, conidia from stock culture were suspended in sterile water and spread on potato-dextrose agar(PDA).After 7 days,newly developed mycelium was transferred to fresh PDA plates and grown for 2 weeks at(25±1)℃with a 12-hr photoperiod under fluorescent light with a photon flux density of 20滋mol/(m2·s).Agar plugs and conidial spore suspensions prepared from the PDA plates used as inoculums.

Herbicides used

Commercial formulation of Paraquat(1,1’-dimethy-4,4’-bipyridinium dichloride,20%a.i.,aqueous solution(AS);Syngenta Crop Protection Company,Switzerland),Fluroxypyr([(4-amino-3,5-dichloro-6-fluoro-2-pyridinyl)oxy]acetic acid,20%a.i., emulsifiable concentrate(EC);Dow AgroSciences company,USA),2,4-D (2,4-dicholrophenoxyacetic acid,57% a.i.,emulsifiable concentrate(EC); Dalian Songliao Chemical Engineering Co.,Ltd,Liaoning,China),Quizalofopp((2R)-2-{4-[(6-chloroquinoxalin-2-yl) oxy]phenoxy}propanoic acid,57%a.i., emulsifiable concentrate(EC);Jinbo Agrochemicals Technology Co.,Ltd, Shangdong,China),Clethodim((E)-2-[1-[[(3-Chloro-2-propenyl)oxy]imino] propyl]-5-{2-(ethyl-thio)propyl}-3-hydroxy-2-cyclohexene-1-one,24%a.i., emulsifiable concentrate(EC);Arysta Lifescience Corporation,Japan),were used in the experiments.

Methods

Herbicide effects on spore germinationStock solutions of herbicides were prepared at recommended field rates and test concentrations were prepared by serial dilution.Suspensions of A.pullulans were mixed at a concentration of 106spores/ml in deionized water with the appropriate concentration of a herbicide resulting in 1X,0.5X,0.2X,0.1X,0.067X,and 0.05X.This corresponded for paraquat and fluroxypyr as 200滋g/ml(1X),100滋g/ml(0.5X),40滋g/ml(0.2X),20滋g/ml (0.1X),13.3滋g/ml(0.067X),and 10滋g/ml(0.05X);for 2,4-D as 570滋g/ml (1X)(field rate),285滋g/ml(0.5X),114滋g/ml(0.2X),57滋g/ml(0.1X),38滋g/ml (0.067X),and 28.5滋g/ml(0.05X);for quizalofop-p as 50滋g/ml(1X)(field rate),25滋g/ml(0.5X),10滋g/ml(0.2X), 5滋g/ml(0.1X),3.3滋g/ml(0.067X),and 2.5滋g/ml(0.05X);for clethodim as 240滋g/ml(1X)(field rate),120滋g/ml (0.5X),48滋g/ml(0.2X),24滋g/ml (0.1X),16滋g/ml(0.067X),and 12滋g/ml (0.05X).A 0.5-ml suspension of spores plus the test herbicides was dislodged evenly over the surface of water agar(1.2%)in 9.0-cm diameter Petri plates with a rubber spatula.Control treatments include spore suspension without herbicide.

The plates were arranged randomly in a single layer and incubated for 12 h at(25±1)℃under light at 20滋mol/(m2·s)photon flux density.Germination percentage was determined by counting one hundred conidia on each of the three replicated plates using a light microscope.Conidia were considered germinated when the germ tube was longer than the spore diameter.In addition,any abnormal development of germ tubes was recorded. Each germination test was conducted at least three times and results averaged.

Herbicide effects on culture-growthIn the growth-inhibition tests,the herbicides were added at above different concentrations to molten,sterilized PDA media.The agar was amended with antibiotics(20 ml/L each of 2.5 mg/ml chloramphenicol and 3.7 mg/ml streptomycin sulphate(Sigma Chemicals,St Louis,MO,USA).One 0.5 cmdiameter agar disc from 14-day-oldculture ofA.pullulanswas then placed in the center of 13-mm Petri dishes, and incubated in a chamber at 25°C with a 12-hr light-and-dark cycle.A control treatment without herbicide was included in each experiment. There were four replicated Petri dishes per herbicide tested.The effect of herbicides on cultural growth was determined from colony diameter measured at 2-day intervals from 2 to 14 days.

Field trialsIn 2011-2012,plots measuring 2 m×4 m,separated by 1.5 m,were planted with approximately 500 presoaked fieldG.aparineL. seeds.Plots were weeded and watered as needed to promote seed germination.Treatments were completely randomized and each treatment had four replications unless stated otherwise.Plots measuring 2×4 m marked with wooden stakes,and mapped for later detection.Applications of treatments were made with 400 ml/plot (500 L/hm2)using the fine mist adjustment of a compressed air sprayer, when winds were calm and just before sundown so that conidia were not immediately expose to direct sunlight. Spore a suspension ofA.pullulanswas prepared as described above, with a conidia alone and as a mixture of conidia and herbicide.Spore suspensions and herbicide were sprayed at the four-to six-leaf growth stage (5.0 to 8.5 cm)of the weeds.Dilution experiments established that a chemical herbicide fluroxypyr at a different doses of 0.2X,0.1X,0.067X,and 0.05X in combination with conidia ofA.pullulansresulted in a synergistic effect.Thein vitrostudy had shown that these rates are the highest concentration of fluroxypyr that allowed spore germination.NL-5H data loggers were set in the field to record temperature,relative humidity,and dew point for the first 72 h after application. Treatments were applied at Xining site on 20 May.Experimental design and treatment in 2012 were the same as in the 2011 trial.

Each year,the effectiveness of all treatments were evaluated at 14 days after inoculation(DAI)for their influence on disease incidence(DI),percent weed control(WC),weed dry weight and wheat yield.DI was determined as the percentage of weeds in each 8 m2area showing disease symptoms.A visual evaluation of WC was taken for the entire 2.0-by-4.0-m treat area.G.aparineL.were evaluated throughout the course of the study and then harvested 14 DAI by cutting the stems at ground level and collecting all aboveground plant tissue.All harvested weed tissue was fully dried for 3 days at 50℃,and then weight for biomass.Wheat yield was obtained by harvesting the whole plot,and wheat seed was weighed and analyzed for moisture content.Wheat seed weight was standardized to 13%moisture.

Statistical analysis

Data on radial growth,spore counts,weed dry weight and weed percent control,wheat yield,and disease incidence,were collected from the experiments and were analyzed using analysis of variance(ANOVA). Data from repeats of the same experiment were pooled when the variances were homogenous.DI,WC,and weed weight data were transformed using square-root transformation prior to analysis to equalize variance.Tukey’s multiple range test(TMRT)(SAS Institute Inc.)was used to compare means of the treatments in each experiment.

Results

Herbicide compatibility study

Interaction between chemical herbicides and fungal spore measured as spore germination after 12 h forA.pullulansin culture were not significantly decreased by fluroxypyr at any labeled concentration of active ingredient but were decreased by 1X(200 mg/ml)(Fig.1).Germination was affected,or only slightly reduced,by fluroxypyr at 0.067X and 0.05X.In contrast,germination ofA.pullulanswith paraquat,2,4-D,quizalofop-p, and clethodim treatment appeared reduced compared with germination ofA.pullulanswith fluroxypyr treatment. Germination was suppressed obviously at concentrations of 1X,0.5X, 0.2X and 0.1X with paraquat,2,4-D, quizalofop-p,and clethodim.

A significant interaction for germ tube length between chemical herbicide andA.pullulanswas observed (Fig.2).Some of the herbicides influenced germ-tube development.The length of hyphae germination fromA.pullulansunder fluroxypyr treatment was longer than hyphae germination under paraquat,2,4-D, quizalofop-p,and clethodim treatment. Abnormal growth features of germ tubes were seen with paraquat,2,4-D, quizalofop-p,and clethodim at 1X, 0.5X,and 0.2X.Paraquat supported the least growth ofA.pullulansconidial,limiting to germ tube length to 7.91滋m.Stunted and shorter germ tubes in comparison with the control were observed with 2,4-D,quizalofop-p,and clethodim at 0.2X.Germ tubes were even shorter at 0.2X with clethodim compared with the control.In case of 2,4-D,germ tubes were generally shorter,with thinner tips compared with a normally grown germ tube.

Differences in radial growth among treatments were observed within 14 days.There was a significant effect of concentration of different chemical herbicides on the radial growth ofA.pullulansin culture(P＜0.05).All concentration of paraquat,2,4-D, quizalofop-p,and clethodim except 0.05X,significantly decreased radial growth ofA.pullulanscompared withits growth on the untreated PDA medium(Fig.3-7).A consistent large reduction of colony growth was observed with 2,4-D,resulting in an atypical and adpressed culture.Mycelial development was sparse and the mycelium took up the green colour of the paraquat solution.

Field trials

In 2011,at Xining site,air temperature within 24 h after application averaged 17℃,with a minimum of 14℃and a high of 26℃.Relative humidity averaged 74%,with a high of 90%and a low of 59%during this time period.In 2012,air temperatures at the Xining site 24 h following application averaged 13℃and range from lows 11℃followed by daytime highs of 25℃for 2 d in a row.Relative humidity at this site averaged 54%,ranging from 49%to 76%during the 24-h period following application.

In 2012,the Xining site received approximately 10.0 mm of rainfall within 24 h after application;in addition,NL-5H loggers suggest that conditions were favorable for dew formation within 2 to 3 h after application, providing 9 to 10 h of possible leaf surface moisture prior to supplemental rainfall.The Xining site dot not receives any recorded rainfall until 48 h after application in 2012.

Across all treatments,significantly higher DI and WC score were observed at Xining site in wheat plots treated with the mixture ofA.pullulanand fluroxypyr(95%and 76 for DI and WC,respectively)compared with plots treated withA.pullulansalone(84% and 65 for DI and WC,respectively) (Table 1).AllA.pullulanstreatments significantly reduced weed biomass compared with the untreated control at 14 DAT at the Xining site.Biomass of weeds from plots treated with the mixture was significantly reduced compared with plots treated with fluroxypyr alone.The greatest reduction in weed dry weight was observed when the mixture of fluroxypyr 20 andA.pullulans.Wheat yield from plots treated by mixture ofA.pullulansand fluroxypyr had significantly higher than the untreated control plots or plots treated with theA.pullulansor fluroxypyr alone.However,in 2012,due to leaf surface moisture condition,performance of all bioherbicide treatments in QAAF site lower than that observed in 2011.

These results indicated a need for the development of formulations to enhance infection in unfavorable environments,particularly when dew periods were not adequate.Thus,further laboratory experiments were conducted and different adjuvants with additional advantages were selected for field tests in the subsequent years.

Discussion

In order for a bioherbicide to be used effectively in weed-control programmes,it should be possible to integrate the biological agent with chemi-cal used in crop production.Any bioherbicide,even if used alone,will have to be resistant to,or tolerant of,herbicides and other pesticides used in the crop production strategy.Hence it is important to define the compatibility ofA.pullulanswith herbicides.

This study was carried out to determine whether selected herbicides adversely affect the activity ofA.pullulans in vitroand on its target,G.aparineL.,and if the susceptibility to theA.pullulansfungus might be increased by herbicide rates.Thein vitrocompatibility assay was used to quantify the effects of the herbicides on spore germination and culture growth.

All herbicides tested were highly toxic to spore germination at the recommended field rate.Charudattan[18]report that there was a concentrationdependend growth inhibition of Cercospora rodmanii Conway,a biocontrol agent forEichhornia crassipes(Mart.)Solms,by 2,4-D.There was a 50%reduction in colony growth by 2,4-D at 200%of the label rate,the highest concentration tested.Interestingly,sporulation of the same fungus was stimulated at concentration of 100,50,25,and 13%of the label rate used.In field studies usingPhoma proboscisHeiny to controlConvolvulus arvensisL.,2,4-D and MCPP were compatible with the fungus at a sublethal rate of 18 g a.i./hm2[19].

Previous work by Wysset al.[16]and Smith and Hallett[15]illustrated that many herbicide reduce the spore production of biocontrol agents but this is the first time paraquat and fluroxypyr have been evaluated as a potential tank mix forA.pullulans.Paraquat, 2,4-D,quizalofop-p,and clethodim appears to reduce growth and stop spore germ tube length ofA.pullulans. However,radial growth ofA.pullulanswas not reduced at the lowest two concentrations of fluroxypyr and germ tube length ofA.pullulanswas similar to the control for all but the 1X,0.5X, 0.2X,and 0.1X.Tank mixing would provide a distinct benefit for use ofA.pullulansas a bioherbicide.However,more work is needed to provide better efficacy with this method.

In this study,temperature and dew period affect disease development in theA.pullulans-fieldG.aparineL.interaction.NL-5H data logger recorded field conditions at Xining site as having rainfall within 24 h after application plus conditions for leaf surface moisture for greater periods of time compared with conditions recorded at Xining site in 2011.Weed weight and WC at Xining site in 2012 were generally less than that observed in 2011.In addition to leaf moisture,this fungal need optimum air temperatures for germination and infection.

Different dose of fluroxypyr in combination with fungus frequently exhibited better weed control compared with fluroxypyr orA.pullulansalone.In most instances,the biocontrol agents and herbicides combinations reduced weed biomass to an extent similar to results observed by Singhet al.and Smith.In Xining in 2011,weed control in the wheat plots by fluroxypyr alone was less than 70% and,even with low Wt,weed control by fluroxypyr was significantly improved by all sequential applications of the bioherbicide treatments at 7 DAT,with most showing an increase in weed control by 14 DAT.Weed control and biomass reduction values reflect the potential of this fungus to be integrated into wheat production.

Differences in weed control and biomass reduction may be explained more readily by environmental differences influencing the degree of pathogen infection in the hours following application.Both years had conditions favoring some dew formation to help provide good leaf surface moisture but Xining site in 2011 air temperature was higher than ideal.Both years had moderate disease incidence,meaning many weeds displayed foliar symptoms;however,the level of DI and weed control was greater at the Xining site in 2011compared with Xining in 2012.Levels of disease may be higher if there is no lag time between application and dew period,where the spores may be less likely to desiccate on the leaf surface.

Table 1Percent disease incidence(DI),weed weight,percent weed control(WC)and wheat yield(kg/hm2)forG.aparinein wheat at Xining after inoculation withA.pullulans,a mixture of both organism and Fluroxypyr a

It has to be mentioned that combinations of certain herbicides and adjuvants can have effects on spore germination that are unlike those resulting from herbicides alone[20].Adjuvants include surfactants,stickers,sun screen agents,humectants,anti-evaporation agents and micro-nutrients[21-26]that may improve bioherbicidal efficacy through various mechanisms.However,we did not test the effects of different combinations of herbicides and adjuvants on the fungus.Therefore, additional studies should be carried out with selected combinations of herbicides and adjuvants used in crop production.

These data provide a guide for the compatibility/incompatibility of A.pullulans with herbicides.This information can be used to develop recommendations for applications of the bioherbicide and chemical herbicides.We believe that the effect herbicides have upon spore germination is more important than on mycelial growth because spores are the source of disease initiation and spread.It is possible that the test chemical is not inhibitory to conidial germination and the germinating mycelium may metabolize and convert the chemical to a fungi-toxic fraction.It is obvious from this study that the effect of chemicals on spore germination do fully correspond with those on already established mycelium on agar plates.Although these results of the in vitro effects of the chemicals may necessarily correlate with their effects under field conditions,they provide a useful guide for selection of appropriate systems for integration of A.pullulans with chemical herbicides.In vitro tests serve as starting points to test for compatibility of different chemicals with biocontrol agents.Generally,in vitro tests provide fairly reliable estimates of compatibility that can be further tested and validated within planta studies.

The results of this research illustrate the potential of this biocontrol agent to be integrated into production systems with other weed management technologies.Continued research on this and other fungal pathogens can benefit and impact weed control practices worldwide by providing needed information and knowledge about the use and application of biocontrol agents.

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Responsible editor:Xiaoxue WANG

Responsible proofreader:Xiaoyan WU

Supported by National Natural Science Foundation of China(No.31160371,30860165), the National Key Technology R&D program of China(No.2012BAD19B02),and the National High Technology Research and Development Program(863Program)of China (No.2011AA10A206).

*Corresponding author.E-mail:liangcheng1979@163.com

Received:May 1,2015 Accepted:June 16,2015