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Research ArticleReceived: 20 July 2012 Revised: 31 August 2012 Accepted article published: 15 October 2012 Published online in Wiley Online Library: 22 November 2012

(wileyonlinelibrary.com) DOI 10.1002/ps.3431

Lime sulfur toxicity to broad mite, to its hostplants and to natural enemiesMadelaine Venzon,a∗ Rafael M Oliveira,b Andre L Perez,b Fredy ARodrıguez-Cruzb and Sebastiao Martins Filhoc

Abstract

BACKGROUND: An acaricidal effect of lime sulfur has not been demonstrated for Polyphagotarsonemus latus. However, limesulfur can cause toxicity to natural enemies and to host plants. In this study, the toxicity of different concentrations of limesulfur to P. latus, to the predatory mite Amblyseius herbicolus and to the predatory insect Chrysoperla externa was evaluated.Additionally, the phytotoxicity of lime sulfur to two P. latus hosts, chili pepper and physic nut plants, was determined.

RESULTS: Lime sulfur at a concentration of 9.5 mL L−1 restrained P. latus population growth. However, this concentrationwas deleterious to natural enemies. The predatory mite A. herbicolus showed a negative value of instantaneous growth rate,and only 50% of the tested larvae of C. externa reached adulthood when exposed to 10 mL L−1. Physic nut had severe injurysymptoms when sprayed with all tested lime sulfur concentrations. For chili pepper plants, no phytoxicity was observed at anytested concentration.

CONCLUSION: Lime sulfur might be used for P. latus control on chili pepper but not on physic nut owing to phytotoxicity. Careshould be taken when using lime sulfur in view of negative effects on natural enemies. Selective lime sulfur concentrationintegrated with other management tactics may provide an effective and sustainable P. latus control on chili pepper.c© 2012 Society of Chemical Industry

Keywords: Polyphagotarsonemus latus; Amblyseius herbicolus; Chrysoperla externa; Capsicum frutescens; Jatropha curcas; calciumpolysulfide

1 INTRODUCTIONThe broad mite Polyphagotarsonemus latus (Banks) (Acari:Tarsonemidae) is a minute mite (0.1–0.3 mm in length) that attacksseveral important crops in tropical and subtropical regions.1,2

Infestations of broad mites are not initially noticeable in fields,because of their small size, and are detected only when mites haveincreased in number and caused serious damage to plants. Broadmites are found in the apical portion of plants, especially in shootstructures, and cause a variety of symptoms on different hosts. Ingeneral, leaves become rigid or bronzed with a shrivelled aspect,and may drop when attack is severe.1 Attacked flowers becomedistorted and fail to open normally. Also, in most attacked hoststhe internodes are greatly shortened, and fruit drop may occurunder severe infestations.2

In Brazil, P. latus is a key pest of several important crops.Among these are chili pepper (Capsicum frutescens L.) and physicnut (Jatropha curcas L.).3,4 The former crop is grown mainly bysmall farmers, and production is mostly destined for the foodprocessing industry. The latter is cultivated for biofuel purpose,and it occupies small as well as large cultivating areas in Brazil.There are no acaricides officially registered in Brazil for broad mitecontrol on either crop. Even though some farmers have appliedpesticides, especially abamectin,5 in an attempt to control thepest, control is mostly unsuccessful, largely because of delayedapplication and the use of incorrect product concentrations.3

With the aim of providing information about an alternativeproduct to control broad mites on these two crops, an evaluation

was made of the toxicity of one of the oldest products usedfor pest control, lime sulfur.6 It consists of a mixture of calciumpolysulfides obtained by boiling calcium hydroxide and sulfur.The toxic effect of lime sulfur on insects and mites is given by thereaction of its compounds, when applied to the plant, with waterand carbon dioxide, resulting in hydrogen sulfide.7 In the past,lime sulfur was intensively used as a winter treatment to controlscales, mites and some diseases, especially in fruiting trees. Bythe 1940s, lime sulfur had been replaced by synthetic organicpesticides.8 Nowadays, with the increase in organic production,the use of lime sulfur has been stimulated by the fact that its useis allowed by several organic certifiers. In Brazil it is currently usedfor fruiting trees, coffee and several other crops, especially thosegrowing under organic cultivation, with the aim of controllingpests and diseases and also for plant nutrition purposes.

∗ Correspondence to: Madelaine Venzon, Agriculture and Livestock ResearchEnterprise of Minas Gerais (EPAMIG), Vila Gianetti 46, 36570– 000, Vicosa,Minas Gerais, Brazil. E-mail: venzon@epamig.ufv.br

a Agriculture and Livestock Research Enterprise of Minas Gerais (EPAMIG), Vicosa,Minas Gerais, Brazil

b Department of Entomology, Federal University of Vicosa, Vicosa, Minas Gerais,Brazil

c Department of Statistics, Federal University of Vicosa, Vicosa, Minas Gerais,Brazil

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The efficiency of lime sulfur has been registered for some

insects9–11 and mite species,12–15 but not yet for P. latus. In spite ofits use, either for pest or disease control, side effects of lime sulfur onnatural enemies have been overlooked, with some exceptions forpredatory mites14,16,17 and a few predatory insects.18,19 Therefore,besides efficiency in controlling P. latus, an evaluation wasalso made of the side effects of lime sulfur on two importantnatural enemies, the predatory mite Amblyseius herbicolus (Chant)(Acari: Phytoseiidae) and the generalist predator Chrysoperlaexterna Hagen (Neuroptera: Chrysopidae). The predatory miteis commonly associated with P. latus in chili pepper plants in Brazil,and its potential as a biological agent for controlling this pest wasrecently reported.20 Chrysoperla externa is a generalist predatoroccurring in several agroecosystems, the adults of which feed onplant-provided food and on honeydew from Hemiptera, and thelarvae of which prey on a variety of soft-body arthropods.21,22

One of the problems related to lime sulfur in some crops is

phytotoxicity.23–25 Thus, field and semi-field experiments (plantsin pots kept under natural conditions) were also carried out toevaluate the toxicity of selected concentrations of lime sulfur tochili pepper and physic nut plants. The aim was to select a limesulfur concentration that would be efficient to control broad miteand selective to natural enemies, and that would not cause toxicityto the crops.

2 MATERIALS AND METHODSRearing and laboratory and semi-field experiments were carriedout at the Experimental Research Station of Agriculture andLivestock Research Enterprise of Minas (EPAMIG) in Vicosa,Minas Gerais, Brazil. Field experiments were carried out at theExperimental Research Station of EPAMIG in Oratorios, MinasGerais.

2.1 RearingThe rearing of P. latus and of A. herbicolus was initiated withmites collected from infested chili pepper plants in the field inthe municipalities of Vicosa and Oratorios. Capsicum frutescenswas established in a greenhouse for P. latus rearing. Potted plantswere kept inside wooden-frame cages (70 × 70 × 70 cm) coveredwith organza screens (90 µm) to isolate the rearing and to avoidcontamination by other arthropods. Rearing of A. herbicolus waskept in arenas of PVC sheets (25 × 12 cm) surrounded by wetcotton to serve as a water source and barrier to prevent miteescape. The sheet was placed over a wet sponge inside a plastictray (30 × 18 × 5 cm) containing water. Pollen of castor bean(Ricinus communis L.) was offered as food on a piece of PVC (4 ×2 cm), and cotton fibres were provided as shelter and ovipositionsite.26 Rearing was done inside climate chambers at 25 ± 2 ◦C, 60± 10% RH and a 14 h photophase.

Adults of C. externa were kept in cages consisting of a PVC tube(8 × 11 cm) covered with nylon gauze and placed on a petri dish(15 cm diameter).22 They were fed with a diet that consisted ofyeast and honey (1:1) offered on a parafilm strip hung inside thecage. Water was provided on a piece of cotton soaked and placedinside a 10 mL vial. Food and water were replaced twice a week.Eggs of C. externa were collected from the cages by cutting theirpedicel and transferring them to glass tubes (2.5 × 8.5 cm). Newlyemerged larvae were fed with an ample supply of eggs of the flourmoth Anagasta kuehniella (Zeller) (Lepidoptera: Pyralidae) untilpupation. The rearing unit was kept at 25 ± 2 ◦C, 70 ± 10% RH and14 h photophase.

2.2 Toxicity of lime sulfur to P. latus and to A. herbicolusThe instantaneous rate of increase (ri) was used as a toxicologicalendpoint.27 This rate is a measure of population increase and iscalculated by the following equation:28

ri = ln (Nf/N0) /�t

where N0 is the initial number of individuals in the population,and Nf is the number of individuals in the population at the endof time interval t. The number of days (t) for the experiment runswas five.

Bioassays were conducted to evaluate the reproduction andsurvival of P. latus and of A. herbicolus when exposed to differentconcentrations of lime sulfur. The arenas used for P. latus consistedof pepper seedlings at 45 days post-emergence. Seedlings wereplaced in individual plastic pots (25 mL) containing soil and manurein a 3:1 proportion. The cotyledonal leaves were removed, and theseedlings remained with two true, fully developed leaves and thecentral shoot leaves. Leaf discs were not used owing to difficultyin keeping P. latus on them.29 For A. herbicolus, arenas consisted ofchili pepper leaf discs (3.0 cm diameter) individually placed insidepetri dishes (3.5 cm diameter). To fix the leaf disc on the petri dish,a water solution of carrageenan (100 g L−1) was prepared, and thedisc was placed before the solution solidification. An attempt touse seedlings as arenas, like those used for P. latus, was made forthe predator, but no predator was recovered after treatment, evenwhen only water was sprayed. In order to minimise the effect ofdifferent arenas, use was made of seedlings at 45 days, when theyhad a total leaf area (true leaves plus shoot leaves equal to 6.3 ±0.47 cm2) more similar to the leaf disc area (7.07 cm2).

Pepper seedlings and leaf discs were sprayed with differentconcentrations of lime sulfur (30 ◦B) (degrees Baume) through aPotter tower30 (Burkard, Rickmansworth, UK). Product sprayingwas carried out under a pressure of 0.34 bar (= 3.44 × 104kPa) and with the application of an equal volume of 2.5 mLper concentration, which corresponded to an aqueous depositof 1.70 ± 0.07 mg cm−2 on the treated area. The tested limesulfur concentrations were 4, 8, 12, 16 and 20 mL L−1. A controltreatment with distilled water was added. Sprayed seedlings andleaf discs were exposed to the environment for 1 h for aqueousspray deposit drying out. Subsequently, ten young females of P.latus, characterised by a transparent body, were placed on eachseedling. For A. herbicolus, ten females (3–4 days old) were placedon each leaf disc, and castor bean pollen was supplied daily.Treated seedlings and leaf discs were kept in a climate chamber(25 ± 2 ◦C, 60 ± 10% RH and 13 h light). The whole pepperseedlings and leaf discs were checked for mite population using astereoscope microscope at 40× magnification. Mite populationswere censused after 5 days by recording the number of immaturesand adults of each species that were alive. Mites were recognisedas dead if they were unable to move for a distance at leastequal to their body length.31 Five replicates were used for eachconcentration for each mite species. Regression analyses wereused to assess the effect of the lime sulfur concentrations on theinstantaneous rate of population increase of P. latus and of A.herbicolus.

2.3 Toxicity of lime sulfur to C. externaChili pepper leaf discs (3.0 cm diameter) individually placed insidepetri dishes (3.5 cm diameter), embedded in a layer of carrageenansolution (100 g L−1), were sprayed with different concentrationsof lime sulfur in a Potter tower, as described above. The tested

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concentrations of lime sulfur were 5, 7.5, 10.0, 12.5, 15.0, 17.5and 20.0 mL L−1. After aqueous spray deposit drying out, onenewly emerged C. externa larva was transferred to each disc.Subsequently, eggs of A. kuehniella were added as food for thelarvae. Larvae were kept on the discs until the beginning of leafdeterioration (6–7 days), and after that they were transferred toglass tubes (2.5 × 8.5 cm) and kept until adult emergence. Foodwas supply every 2 days, and mortality was daily assessed. Tenreplicates were done for each tested concentration. Each replicateconsisted of one C. externa larva on one treated leaf disc. Survivalcurves were estimated by the Kaplan–Meier technique32 andcompared using the log rank test.33 The Bonferroni method wasused to correct the significance level for multiple comparisons.34

2.4 PhytotoxicityA field experiment was carried out to evaluate the toxicity of limesulfur concentrations to chili pepper plants. The experiment wascarried out in Oratorios, from 13 to 21 May 2009. Chili plants werein fructification stage, and they were 1 m spaced. Spacing betweenrows was 1.2 m. The tested concentrations of lime sulfur were 0,5, 10, 15 and 20 mL L−1. All five treatments were randomised inblocks with four replicates. Each plot consisted of three rows withfive plants, but data were collected from the three central plants.

For physic nut plants, a semi-field experiment was carriedout from 1 to 15 February 2010 in Vicosa. Potted physic nutplants (2 months old) were kept in natural conditions. The testedconcentrations of lime sulfur were 0, 5, 10, 15 and 20 mL L−1. Arandomised design was used, and treatments and control werereplicated 5 times.

In both experiments, plants were sprayed with the tested limesulfur concentrations using a costal sprayer Brudden SS modelwith a capacity of 5 L. Visual symptoms of phytotoxicity for eachconcentration tested were evaluated by two observers after 1,5 and 10 days of treatment. A visual scale of phytotoxicity wasused:35 rank 0: plants with normal leaves and no signs of burns;rank 1: plants with slightly damaged leaves and/or with smallburned areas; rank 2: plants with medium damaged leaves, yellowwith burnt edges and tips; rank 3: plants with heavily damagedleaves, showing severe defoliation.

Phytotoxicity data were analysed under a split-plot design, thefactors of which were concentration (mL L−1) and time (days). Asa significant interaction between these factors was verified, theconcentration levels were studied by the fit of linear regressionmodels at each time. To compare the models, an F-test wasperformed for model identity.36 These analyses were carried outusing R software.37

3 RESULTSThe instantaneous rate of increase (ri) of P. latus linearly decreasedwith increasing lime sulfur concentration (Fig. 1). This rate wasnull, indicating that the population was stable and did not growat a concentration of 9.5 mL L−1.

Amblyseius herbicolus also showed a linear decline in populationgrowth with increasing lime sulfur concentration, but it wasaffected at a lower concentration than its prey P. latus. Theconcentration restraining predatory mite population growth (ri

= 0) was obtained when predators were exposed to 7.4 mL L−1 oflime sulfur.

The survival of C. externa varied with tested lime sulfurconcentration (Fig. 2). Predators that survived for 20 days after

Figure 1. Instantaneous rate of increase (ri , day−1) of Polyphagotarsonemuslatus (open triangles; y = 0.018 − 0.1718x, dferror = 28, F = 151.13, P <

0.0001, R2 = 0.83) and of Amblyseius herbicolus (open circles; y = 0.0275 −0.2047x, dferror = 28, F = 252.39, P < 0.0001, R2 = 0.90) exposed to increasedconcentrations of lime sulfur.

Figure 2. Survival distribution curves for Chrysoperla externa exposed todifferent lime sulfur concentrations.

exposure reached adulthood. The survival of predators exposedto concentrations up to 12.5 mL L−1 did not differ from the controlwhen only water was applied (P > 0.05) (Table 1). Predatorsexposed to the highest tested concentrations (15–20 mL L−1) hadsignificantly lower survival compared with the control treatment(Fig. 2). At a concentration of 10.0 mL L−1, 50% of the testedpredators reached adulthood.

As regards phytotoxicity, chili pepper sprayed with lime sulfurhad no symptoms of injury. Even the highest tested concentration(20 mL L−1) did not cause toxicity to chili pepper plants in thefield. However, for physic nut the opposite was found. The analysisof variance under a split-plot design showed significant effects ofconcentration (F4,40 = 25.68, P < 0.001), of time (F2,40 = 13.80, P <

0.001) and of time and concentration interaction (F8,40 = 3.63, P <

0.001). Plants had injury symptoms at all treated concentrations.The severity of symptoms increased linearly with increasing limesulfur concentration (Fig. 3). The model identity test showed thatthe injury also increased with time after application (F4,69 = 4.28,P = 0.0038). After 5 days, phytotoxicity ranks were higher thanthose after 1 day (F2,69 = 6.39, P = 0.0028), but similar to those

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Table 1. Pairwise comparison of the different concentrations of lime sulfur (mL L−1) for survival of Chrysoperla externa. χ2 (Log rank test) and Pvaluesa

Lime sulfur concentrations χ2 P Lime sulfur concentrations χ2 P Lime sulfur concentrations χ2 P

0 × 5.0 1.1 0.2920 5.0 × 15.0 7.4 0.0067 10.0 × 17.5 3.3 0.0674

0 × 7.5 4.8 0.0289 5.0 × 17.5 10.5 0.0012* 10.0 × 20.0 4.4 0.0362

0 × 10 6.2 0.0125 5.0 × 20.0 12.8 0.0003* 12.5 × 15.0 0.7 0.4030

0 × 12.5 6.3 0.0120 7.5 × 10.0 0.8 0.3620 12.5 × 17.5 2.5 0.1130

0 × 15.0 10.6 0.0011* 7.5 × 12.5 0.8 0.3700 12.5 × 20.0 2.5 0.1130

0 × 17.5 13.4 0.0002* 7.5 × 15.0 4.4 0.0366 15.0 × 17.5 0.9 0.3470

0 × 20.0 16.5 <0.0001* 7.5 × 17.5 7.6 0.0059 15.0 × 20.0 0.8 0.3610

5.0 × 7.5 1.6 0.2120 7.5 × 20.0 10.1 0.0015* 17.5 × 20.0 0.0 0.9680

5.0 × 10.0 3.3 0.0683 10.0 × 12.5 0.1 0.7500

5.0 × 12.5 3.5 0.0612 10.0 × 15.0 1.5 0.2190

*aSignificant by the log rank test with Bonferroni correction (P < 0.0018).

Figure 3. Toxicity of lime sulfur concentration to physic nut plants 1, 5and 10 days after product spraying. Time 1 (y = 0.04 + 0.07x, dferror = 23,F = 24.77, P < 0.0001, R2 = 0.80); time 5 (y = 0.10 + 0.09x, dferror = 23, F =105.192, P < 0.0001, R2 = 0.98); time 10 (y = 0.18 + 0.09x, dferror =23, F =74.71, P < 0.0001, R2 = 0.93). Rank 0: normal leaves and no signs of burns;rank 1: slightly damaged leaves and/or with small burned areas; rank 2:medium damaged leaves, yellow with burnt edges and tips; rank 3: heavilydamaged leaves, showing severe defoliation.

after 10 days (F2,69 = 0.09, P = 0.9133). Phytotoxicity ranks weresignificantly higher after 10 days than after 1 day (F2,69 = 6.35, P =0.0029). At higher concentrations, leaves had small burned areasand tips were damaged, but no defoliation was observed.

4 DISCUSSIONPopulation growth of P. latus was curbed (ri = 0) when it wasexposed to a concentration of lime sulfur of 9.5 mL L−1. Abovethis concentration, the population of P. latus declined towardsextinction. The use of lime sulfur at 6 mL L−1, which correspondedto null population growth for Tetranychus evansi Baker & Pritchard,was sufficient to control this mite population on tomato plantsgrown in the greenhouse.15 The concentration of lime sulfurthat was found to stop population growth of P. latus was belowcommon field rates applied by farmers in most crops, which ranged

from 20 to 40 mL L−1 (29–32 ◦B).17 Although the P. latus populationmight be controlled when using lime sulfur at 9.4 mL L−1, it wasfound that at this concentration the instantaneous grow rate ofpredatory mite A. herbicolus showed negative values (ri = −0.06day−1), indicating a decline in its population towards extinction.

Predatory mite population growth was negatively affected at alower concentration than its prey. This may have occurred becauseP. latus has a higher reproductive potential than A. herbicolus,4,20

which can minimise mortality caused by lime sulfur. Populationcompensation was used to explain the greater reproductive out-put of surviving individuals exposed to sublethal concentrations ofbiocides.27 Thus, the A. herbicolus population can be affected neg-atively by a lower concentration of lime sulfur than its prey P. latus.

A similar response was observed for other predator–preysystems when sulfur was applied.31 Based on LC50 estimates, thepredatory mite Iphiseiodes zuluagai Denmark & Muma was 17 timesmore tolerant to sulfur than its prey, the phytophagous southernred mite Oligonychus ilicis (McGregor). However, exposure to sulfurLC25 led to predator extinction after 7 days of exposure, while,for the prey, extinction was not reached with a concentration ashigh as LC90. According to the authors, this might be explainedby the higher population compensation of O. ilicis, which is dueto its higher reproductive potential, than I. zuluagai. Populationcompensation might occur because survivors of sulfur exposurewill have greater resource availability and are likely to reproduceat a higher rate, minimising the acaricidal effect at the populationlevel.31

The survival of C. externa decreased with increasing lime sulfurconcentration, and a significant reduction was observed wheninsects were exposed to a lime sulfur concentration above 15mL L−1. At the maximum concentration tested (20 mL L−1), only10% of larvae reached adulthood. At 10 mL L−1, which is nearto the concentration that proved to be efficient in stopping thepopulation growth of P. latus (9.5 mL L−1), survival of C. externawas not significantly different from that of the control, but only50% reached adulthood. There are few reports on the side effectsof lime sulfur on predatory insects.18 The mortality of larvae of twoCoccinellidae (Stethorus punctum picipes and Harmonia axyridis)after 48 h of exposure of lime sulfur to the field rate and to halfthe field rate for a hop crop was null for S. punctum picipes andonly 6.7% for H. axyridis.18 Based on this lethal toxicity evaluation,the authors concluded that the product had low toxicity to theevaluated predators. However, in the present experiments, theimportance of evaluating the sublethal toxicity of products was

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shown. A negative effect of increase in the lime sulfur concentrationon C. externa survival over time was observed. In fact, reductionin survival did not begin until 5 days after lime sulfur exposure. Ifthe experiment had been stopped before this time, it would havebeen concluded that, even at a higher lime sulfur concentration,the product was safe for the predator.

Phytoxicity of lime sulfur has been reported for some crops,but not yet for physic nut. Plants showed injury symptoms whensprayed with all tested concentrations of lime sulfur. Plants sprayedwith lime sulfur at a concentration near to that curbing P. latuspopulation growth (9.5 mL L−1) had severe toxicity symptoms,which would impede the use of lime sulfur for P. latus control inphysic nut. However, for chili pepper plants, no phytoxicity wasobserved, even at higher tested concentrations. The componentthat is believed to cause plant injury is the soluble sulfide oflime sulfur, which acts by reducing carbon dioxide assimilation.25

However, it is not clear yet why some plant species are moresusceptible than others.

The use of lime sulfur as an alternative for controlling P. latus ispossible in chili pepper plants, but not in physic nut on accountof product phytotoxicity. The negative side effects on naturalenemies, as shown here, demonstrated that care must be takenwhen using such a product. One possibility that remains to betested is the combined use of a selective lime sulfur concentration,for instance 5 mL L−1, and natural control provided by predatorsin the field. This strategy might be successful when other tacticsfor improving biological control are also employed.

ACKNOWLEDGEMENTSThe National Council of Scientific and Technological Development(CNPq) and Minas Gerais State Foundation for Research Aid(FAPEMIG) are thanked for financial support and for fellowshipsawarded to the authors. The authors also thank Mineracao LapaVermelha for providing lime for lime sulfur preparation.

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