influence of herbicides on cellulolytic, proteolytic and phosphate solubilising · pdf...

Influence of herbicides on cellulolytic, proteolytic and phosphate solubilisingbacteriaP.C. LATHA AND H. GOPAL

International Journal of Plant Protection (April, 2010), Vol. 3 No. 1 : 83-88

Key words :Herbicides,Cellulolytic,Proteolytic,Phosphatesolubilisingbacteria

Accepted :February, 2010

The present day agriculture depends uponhigh yielding varieties, inorganic fertilizers

and pesticides to achieve the increased foodproduction required to keep pace with theincreasing population. The progressivemodernization of irrigated rice cultivation inIndia, using the above technologies has led totremendous increase in rice production, whichhas more than doubled over the last 35 years,mainly driven by 85% increase in productivity.

In India, herbicides constituted only 15percent of the total consumption of pesticides,compared to the worldwide consumption of47.5 per cent. The herbicide consumption isexpected to increase dramatically in future asthe use of herbicides has been expanding morerapidly than that of the other pesticides (Bhanand Mishra, 2001). Herbicide usage, which wasearlier confined to plantation crops, has nowexpanded to crops like wheat (42 per cent ofthe total consumption of herbicides) and rice(30 per cent) with the states of Punjab, UttarPradesh, Tamil Nadu and Andhra Pradeshleading in the consumption of more herbicides.

Since the herbicides are used when thecrop is either absent as pre-emergence or atits early stage of growth as post-emergence, ahigh proportion of the herbicide reaches the soiland accumulates in the microbiologically active

HIND AGRICULTURAL RESEARCH AND TRAINING INSTITUTE

top layer of 0-15 cm soil. Herbicides beingbiologically active compounds, an unintendedconsequence of the application of herbicidescould influence the microbial ecological balancein the soil leading to significant changes in thepopulations of microorganisms and theiractivities and affecting the productivity of soils(Boldt and Jacobsen, 1998). Hence, theincreasing reliance of rice cultivation onherbicides has led to concern about theirecotoxicological behaviour in the rice fieldenvironment.

With this background, the presentinvestigation was carried out with the mainobjective, to understand and predict the effectof herbicides viz., 2,4-D-2ethylhexyl ester (2,4-DEE), butachlor, pretilachlor and pyrazosulfuronethyl on rice soil microorganisms and theiractivities, which could lead to their judicioususe and thereby to reduce their negative effects,if any on the environment.

MATERIALS AND METHODSA laboratory incubation experiment was

conducted using field soil obtained fromwetlands of TNAU, Coimbatore, by devisingmicrocosms to study the effect of differentconcentrations of herbicide formulations oncultivable microflora and potential enzyme

See end of the article forauthors’ affiliations

Correspondence to :P.C. LATHADepartment ofAgriculturalMicrobiology, TamilNadu AgriculturalUniversity,COIMBATORE (T.N.)INDIA

SUMMARYExperiments were conducted at the Department of Agricultural Microbiology to study the effect ofherbicides viz., 2,4-DEE, butachlor, pretilachlor and pyrazosulfuron ethyl on soil microorganisms andenzyme activities in laboratory microcosms, in vitro effect on growth of pure cultures of Azospirillumlipoferum and Bacillus megaterium and their nitrogen fixation and phosphate solubilisation abilitiesand also on the effect of herbicide application to rice in pot culture inoculated with biofertiliser, Azophos.Soil microbiological, biochemical, chemical and agronomic variables were also studied in a permanentherbicide trial to study the impact of long term herbicide application in transplanted low land rice-ricesystem.In laboratory incubation studies, it was observed that butachlor was more inhibitory to microbialpopulations (7.85 to 34.20% reduction over control) and soil enzyme activities (5.03 to 19.11% reductionover control) when compared to 2,4-DEE, pretilachlor and pyrazosulfuron ethyl. Among the herbicidestested, the soil microbial population and enzyme activity inhibition followed a trend, butachlor > 2,4-DEE> pretilachlor > pyrazosulfuron ethyl.

Research Paper :

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HIND AGRICULTURAL RESEARCH AND TRAINING INSTITUTE[Internat. J. Plant Protec., 3 (1) April, 2010]

activities.

Soil microcosms :Soil obtained from wetlands of TNAU, Coimbatore

was air dried and passed through 2 mm sieve. The soil inportions equivalent to 250 g oven dry weight was placedin 500 ml beakers, adjusted to the required level ofmoisture in flooded condition and pre-incubated at 30 ±1oC for 3 days for conditioning. Appropriate quantities ofthe herbicide formulations were added to the soil tomaintain concentrations of herbicides at control, FR (fieldrate), 2FR (2 x field rate), 5FR (5 x field rate), 10 FR (10x field rate) and 100 FR. Soil without herbicide applicationwas also maintained as control. The field rates ofapplication for different herbicides were 0.75 kg ai. ha-1

for 2,4-DEE, 1.0 kg ai. ha-1 for butachlor, 0.30 kg ai. ha-1

for pretilachlor and 25g ai. ha-1 for pyrazosulfuron ethyl.Water was adjusted to the same levels in all the treatmentsincluding control. A 3 cm depth of overlying water wasmaintained in all the treatments. The treated soils werethen covered with plastic sheets having small holes andincubated at 30 ± 10C in the dark for 30 days. For samplingthe soil at different intervals, overlying water was carefullyremoved and the surface soil was collected from a depthof 0-3cm using a spatula (Saeki and Toyata, 2004). Sampleswere drawn at 0, 7, 15 and 30 days after application ofherbicides and analysed for the effect of herbicides on soilmicrobial populations and enzyme activities.

Enumeration of cellulolytic, proteolytic and phosphatesolubilising bacteria :

The population of cellulase producing bacteria wasdetermined by plating soil dilutions on CMC agar medium(Henriksen and Breland, 1999). The plates were incubatedin the dark at 25 ± 10C for 5 days and then flooded withcongo red (0.1% solution, 15 minutes), followed by theaddition of 1M NaCl and 1M HCl for visualization ofsolubilization zones around the bacterial colonies (Teatherand Wood, 1982).

Viable counts of protein degrading bacteria in soilwere enumerated on skim milk agar medium (Aneja, 1996).The colonies that showed zones of solubilisation of caseinwere recorded as proteolytic bacteria.

The population of phosphate solubilising bacteriapresent in the soil samples was enumerated on sucrose –tri calcium phosphate agar medium (Pikovskaya, 1948).The bacterial colonies surrounded by clear zones wererecorded as phosphate solubilizers.

RESULTS AND DISCUSSIONThe effect of herbicides, 2,4-DEE, butachlor,

pretilachlor and pyrazosulfuron ethyl at differentconcentrations and at various incubation times werestudied using laboratory microcosms set up in theDepartment of Agricultural Microbiology.

The herbicides 2,4-DEE, butachlor, pretilachlor andpyrazosulfuron ethyl were applied at differentconcentrations to soil microcosms and the resultantdifferences in the population of cellulolytic bacteria weremonitored (Table 1and Fig. 1). A comparison of the threefactors revealed that butachlor among herbicides, 100FRamong the concentrations and 15 days after applicationrecorded significantly lower population (4.988, 4.900 and5.027 log CFU g-1 soil, respectively). Cellulolytic bacterialpopulation were significantly influenced by the interactiveeffects of herbicide x concentration. The highestpopulation was observed in the control treatment of allherbicides and was comparable to the population in 1FRpretilachlor, 1FR pyrazosulfuron and 2FR pyrazosulfuronethyl. Non significant effects were observed for herbicidex days and herbicide x concentration x days interaction.

Significant differences in the numbers of proteolyticbacterial population were registered in the presence ofdifferent concentrations of the herbicides whenenumerated at 7, 15 and 30 days after herbicide application(Table 2). Significantly lower population (4.362 log CFUg-1 soil) was observed in the presence of butachlorcompared to 2,4-DEE (4.395 log CFU g-1 soil), pretilachlor(4.421 log CFU g-1 soil) and pyrazosulfuron ethyl (4.427log CFU g-1 soil). Higher concentrations of the herbicideswere observed to lower the population of proteolyticbacteria, the 100 FR treatment recording the lowestpopulation (4.321 log CFU g-1 soil). The influence of theinteraction between herbicides and concentrationsignificantly influenced the proteolytic bacterial

P.C. LATHA AND H. GOPAL

3.5

4

4.5

5

5.5

6

6.5

log C

FU

g-1 s

oil

T1 T2 T3 T4 T5 T6

Treatments

Cellulolytic bacteria Proteolytic bacteria P solubilising bacteria

Fig. 1 : Impact of herbicide application on populations ofcellulolytic, proteolytic and P solubilizing bacteriain the long term herbicidal trial

Cellulolytic bacteria Protedytic bacteria Psolubilising bacteria

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populations. Butachlor at 100 FR had the lowestpopulation (4.205 log CFU g-1 soil), while thehighest population was observed in control and1FR treatments of all herbicides.

Changes in the phosphate solubilizingbacterial population as a consequence of theapplication of herbicides at different concentrationswere monitored at different days and arepresented in Table 3. Among the herbicides, thelowest population was observed in butachlor (4.573log CFU g-1 soil) followed by 2,4-DEE (4.591 logCFU g-1 soil), pretilachlor (4.599 log CFU g-1 soil)and pyrazosulfuron ethyl (4.609 log CFU g-1 soil).The highest concentration of herbicides (100FR)recorded the lowest population of 4.547 log CFUg-1 soil, compared to the other concentrations. Nonsignificant interaction effects of the factors wereobserved for phosphate solubilising bacterialpopulations.

Weeds are the major biological constraint inmost rice growing areas of the world. Unlike theperiodic outbreaks of insect pests and plantdiseases, weeds are ever present and threatening.Problems associated with weeds in rice cultivationare mounting dramatically in South and SoutheastAsia because of the reduced availability ofaffordable labour. The lack of suitable weedcontrol alternatives has led to the increasingreliance on herbicides in many rice growing areasand their use is increasing as they are lessexpensive and convenient than manual labour, veryeffective and easy to use. In India, herbicideconsumption is expected to increase in future asthe use of herbicides has been expanding morerapidly than that of the other pesticides used inpest and disease control (Bhan and Mishra, 2001).

Herbicides may affect non target organismsincluding microorganisms. Herbicide inducedchanges in abundance, diversity and activity of soilmicrobial communities may in turn influencemicroorganism mediated processes that areimportant to sustainable agriculture like recyclingof plant nutrients. This is especially true of riceecosystem which is composed of extremely largenumber of very diverse microbial subhabitats inspace and time, and where productivity andenvironmental quality are both linked tomicroorganisms that are the main carriers ofbiocatalytic functions affecting nutrient supply tothe crop and is viewed as a major supporter of thesystem’s sustainability (Reichardt et al., 1998).

INFLUENCE OF HERBICIDES ON CELLULOLYTIC, PROTEOLYTIC & PHOSPHATE SOLUBILISING BACTERIA

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Hence, there are concerns about thedeleterious effects of herbicides on the riceecosystems and such impacts cannot beoverlooked when increasing attention is beingfocused on environmental issues.

The measurement of the microbialpopulation and processes at a large numberof concentrations is a better way to study thetoxic effects of pesticides (Van Beelen andDoelman, 1997). The 10FR treatment isrecommended in laboratory tests to assess theside effects of pesticides on soil microflora(Sommerville, 1987). Investigations performedwith high concentrations like 100 times higherthan field rates although not immediatelyrelevant for agricultural management practice,may be useful for assessing environmental riskin those soils where monocultural practicesrequire repeated application of the herbicide.Such positive control treatment which isalready known to have an effect on microbescan also be used to validate the sensitivity ofvarious measurements (Ahtiainen et al.,2003).

The monitoring period is a most importantpart for the assessment of pesticide effects anda minimum of 30 days has been recommendedfor the recognition of persistent effects on soils.A delay of 30 days in the restitution of normality(recovery period) after herbicide applicationshould be considered normal with ecologicalconsequences being negligible, a delay of 60days is not unusual, and the ecologicalconsequences are tolerable and a delay ofgreater than 60 days is unusual with ecologicalconsequences which may eventually be critical(Domsch et al., 1983).

The results of this experiment revealedthat the application of herbicides reduced thepopulation of all the bacteria enumerated duringthe study with butachlor showing highestreduction in the populations. This effect wasstronger with increasing concentration of theherbicides employed. However, the populationsat 1FR (and also 2FR for pyrazosulfuron ethyl)concentrations recovered within 30 days toreach populations not significantly differentfrom the control treatments.

Debnath et al. (2002) have reported thatbutachlor at 2 kg ai. ha-1 (equivalent to 1 µg g-

1soil) stimulated the population of phosphate


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solubilising bacteria though a contrasting effectwas observed in the present experiment. Areduction in the cellulose degradation wasreported by the application of butachlor at 16µg g-1soil after four weeks of application(Katayama and Kuwatsuka, 1991) which wasalso observed in this experiment as a significantreduction in the population of cellulolyticbacteria at 100FR concentrations.

It was observed in this experiment that thedetracting effects of herbicides towards allbacteria and enzyme activities decreased withtime. Recovery of microbial populations andenzyme activities after initial inhibition may bedue to microbial adaptation or most probablydue to their degradation (Ismail et al., 1998).2,4-DEE with a half life of 18 days (Jayakumarand Ramulu, 1993) chloroacetamide herbicideslike butachlor (half life of 19 days) andpretilachlor (17 days) degrade quickly in soil(Sahid and Yap, 1994, and Murato et al., 2004).Both Gigliotti et al. (1998) and Saeki and Toyota(2004) have reported non-significant effects ofthe sulfonyl urea herbicide bensulfuron methylon bacterial populations due to the fact thatsulfonyl urea herbicides degrade rapidly in thesoil. Pyrazosulfuron ethyl, the sulfonyl ureaherbicide used in this study had been alsoreported to have half life of 16 days in anexperiment conducted in Taiwan (Chu et al.,2002). The initial decrease followed by smallincreases in populations could also be due tomicrobial multiplication on the increased supplyof nutrients available in the form ofmicroorganisms killed by herbicides (Boldt andJacobsen, 1998).

Authors’ affiliations:H. GOPAL, Department AgriculturalMicrobiology, Tamil Nadu Agricultural University,COIMBATORE (T.N.) INDIA

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