Effect of Crude Plant Extracts onMushroomMite , Luciaphorus sp . ( Acari : Pygmephoridae )

The use of plant extracts for controlling agricultural pests has become increasingly popular in the recent years. Mushroom mite, Luciaphorus sp., is a destructive pest of several mushroom species and has been reported to cause severe loss of mushroom cultivation in many settings. The efficacies of 23 rhizome and leaf extracts were evaluated against female adults of Luciaphorus sp. At 3 days after treatment, the rhizome extracts derived from Curcuma xanthorrhiza Roxb. and Zingiber montanum (Koenig) Link ex Dietr. were found to have very strong acaricidal activities, resulting in 100% mite mortality, followed by Curcuma longa Linn. (98.89%), Zingiber zerumbet (L.) Smith. (97.78%), Kaempferia parviflora Wall. Ex Baker (88.89%), and Zingiber officinale Roscoe. (84.44%). The leaf extracts of Ocimum sanctum Linn. and Melissa officinalis L. also resulted in 100% mite mortality 3 days after treatment, while the other leaf extracts induced mite mortality only below 70%. The results suggested that rhizome extracts of C. xanthorrhiza and Z. montanum and leaf extracts of O. sanctum and M. officinalis have a great potential for future development as natural acaricides for controlling Luciaphorus sp.


Introduction
Luciaphorus sp.(Acari: Pygmephoridae) is considered as one of the most destructive pests of mushroom cultivation in Thailand.This pygmephorid mite is responsible for the severe production losses of Lentinus squarrosulus (Mont.)Singer, L. polychrous Lev., Auricularia auricula-judae (Bull.:Fr.)Wettst.and Flammulina velutipes (Curt.:Fr.)Karst.mushrooms in the Northeast of Thailand [1].Despite that, little is known about the effective measures for controlling this mite and routine horticultural hygiene is the only procedure to alleviate the problem.To make the situation worse, desperate mushroom growers in Thailand use a large amount of carbamate and organophosphate insecticides and even some harmful solvents to manage this mite; however, this results in very limited success [2].
As a consequence, this mite becomes rapidly resistant and more harmful miticides have to be applied.The use of toxic miticides raises the concerns because of their effects on environments, human safety, and nontarget organisms.Hence, the use of nontoxic natural products for controlling this agricultural pest has been proposed.There are several higher plants that are rich in natural substances, especially the secondary metabolites, such as terpenes, steroids, alkaloids, phenolics, and cardiac glycosides, and can be used as nonharmful, environmentally friendly agents for insect control.Indeed, the use of natural compounds derived from plant extracts has been suggested as alternative treatments for insect and mite controls due to their multiple modes of action, including repellence, feeding and oviposition deterrence, toxicity, and growth regulatory activity [3][4][5][6].Moreover, plant-based pesticides are often found to contain a mixture of active substances which can delay or prevent resistance development [7].Therefore, in this study, the acaricidal activities of 23 plant extracts were determined against the mushroom mite, Luciaphorus sp.

Mushroom and Mite Culture. Lentinus squarrosulus
Mont. mushroom culture was obtained from the Mushroom Growers Society of Thailand.The mycelium was freshly sub cultured on 90 mm plastic Petri dish plates containing potato dextrose agar (PDA, Sigma) and grown at 25 • C.
Luciaphorus sp.mites were collected from infested L. squarrosulus composts obtained from Rapeephan mushroom farm in Khon Kaen province in the Northeast of Thailand.A pair of male and female mites was maintained at 28 • C using fresh L. squarrosulus spawn that was grown on sawdust and sorghum grains in a glass bottle.The offspring that were in-house bred inside this glass bottle were used for all the experiments.

Preparation of Plant Extracts.
Leaves and rhizomes of 23 plants were collected locally from Mahasarakham province in the Northeast of Thailand (Table 1).Plant materials were cut into small pieces and dried in hot air oven at 45 • C for 3 days.
The dried plants were separately ground into powders using a small grinder and stored at 4 • C in polypropylene bags.For extraction, 100 g of each powdered plant materials and 300 mL of 80% ethyl alcohol were added into sterile 2L Erlenmeyer flask, and the flask was agitated at 100 rpm for 24 h.After filtering through a Buchner funnel and Whatman No. 1 filter paper, the extracts were concentrated under low pressure using rotary evaporator.The crude extracts were reconstituted to have the concentration of 20% (w/v) using 80% ethyl alcohol (v/v, in distilled water) and stored at 4 • C in glass vials to be used as stock plant extracts.For the tests, these stock plant extracts were dissolved in distilled water containing 0.05% Tween 80 to have the concentration of 5% (w/v).

Bioassay.
For evaluation of each plant extract, 100 adult female mites were transferred to a 50 mm Petri dish plate containing mushroom mycelial culture grown on PDA medium, and the plate was then sprayed with 500 µL of each plant extracts prepared at the concentration of 5% (w/v).The same volumes of the sterilized distilled water (DW) and 0.005% Omite (commercial miticide) were used as control groups.The experiments for each plant extracts were performed in triplicates.All plates were incubated in the growth chamber at 28 • C and 85% RH in the dark.The mortality of mites was recorded every day for 5 days after application with plant extracts.

Statistical Analysis.
Data on the percentages of mite mortality due to application with plant extracts were arcsinetransformed and subjected to analysis of variance using the general linear models procedure (SAS Institute, Cary, NC, USA).Significant differences between the treatments were determined using the LSD test at P < 0.05.

Acaricidal Activity of Leaf Extracts.
The levels of mite mortality after applications with leaf extracts also reached maximum on day 3 (Figure 2).On day 3, the leaf extracts of O. sanctum and M. officinalis resulted in maximum mortality (100%), but the other treatments were shown to result in mortality at levels below 70% (Figure 2).This was not unexpected because only the applications with the leaf extracts of O. sanctum and M. officinalis caused over 70% of mortality on day 1 (data not shown).Also, on day 2, mortality rates in all treatments increased and the leaf extracts of O. sanctum and M. officinalis still resulted in mite mortality at the levels significantly higher than the rest, Psyche 3

Discussion
Several plants have been found to contain bioactive compounds with a variety of biological actions against insects and mites, including repellent, antifeedant, antiovipositional, toxic, chemosterilant, and growth regulatory activities [4,8].Therefore, botanical insecticides have long been recommended as attractive alternatives to synthetic chemical insecticides for pest management because these chemicals pose little threat to the environment or to human health [9].For example, the crude foliar extracts of five subfamilies of Australian Lamiaceae, including Ajugoideae, Scutellarioideae, Chloanthoideae, Viticoideae, and Nepetoideae, were found to have contact toxicity against the polyphagous mite (Tetranychus urticae Koch) [10].This T. urticae could also be inhibited by the essential oil in crude foliar extract of sandalwood (Santalum austrocaledonicum), resulting in 87.2 ± 2.9% mortality and 89.3% reduction of the total number of eggs on leaf disks treated with this oil [11].Piperoctadecalidine, which is the alkaloid isolated from Piper longum Linn., was also found to have activities against T. urticae at LD 50 of 246 ppm [12].Moreover, Aslan et al. [13] reported that essential oil vapours from Satureja hortensis Linn., Ocimum basilicum Linn, and Thymus vulgaris Linn.had potential against T. urticae, but the essential oil obtained from S. hortensis was the most effective at 1.563 µL/L air dose by causing 100% mortality of T. urticae after 4 days of treatment.
In recent years, many studies have also been conducted to investigate the activities of plant extracts or essential oils against carmine spider mite (Tetranychus cinnabarinus Boisd.Tunc) and Hawthorn red spider mite (Tetranychus viennensis Zacher).The chloroform extract of Kochia scoparia Linn.was shown to have rapid acaricidal activities against T. urticae, T. cinnabarinus, and T. viennensis, resulting in the highest mortality at 92.58, 88.88, and 84.47%, respectively, within 24 h after treatment [14].Also, toxicity against T. cinnabarinus and T. viennensis could be quickly induced by the petroleum ether extract of Juglans regia Linn., resulting in mortality rates at 81.58 and 78.58%, respectively, within 24 h [7].
In this study, the rhizome extracts of C. xanthorrhiza and Z. montanum and the leaf extracts of O. sanctum and M. officinalis at the dose of 5% (w/v) were found to be highly effective against female adults of Luciaphorus sp.The results revealed that the rhizome extracts were likely to have more potent acaricidal activities than those derived from leaves.The acaricidal activities of plant extracts against Luciaphorus sp.mites have been previously described.The essential oils derived from lemon grass (Cymbopogon citratus Stapf.) and citronella grass (Cymbopogon nardus Rendle) were shown to be effective against Luciaphorus perniciosus Rack., and the median effective concentration (EC 50 ) was 18.15 and 19.66 ppm, respectively [26].In addition, the essential oils of Litsea cubeba Pers.were effective against L. perniciosus by contact and fumigation methods with LD 50 values equivalent to 0.932 and 0.166 ppm, respectively [27].
In conclusion, the results in this study suggest the possibility of developing plant extracts derived from the rhizomes of C. xanthorrhiza and Z. montanum and the leaves of O. sanctum and M. officinalis for controlling Luciaphorus mites.The effective concentration and mode of action of these plant extracts against Luciaphorus sp.remain to be determined for the future development of highly potent products to be used in the real settings.

Figure 1 :
Figure 1: The mortality rates of adult female Luciaphorus sp. after being treated with 5% rhizome extracts at 3 days after application.Bars (mean ± SE) with same letter(s) are not significantly different as determined by LSD test at P < 0.05.

Figure 2 :
Figure 2: The mortality rates of adult female Luciaphorus sp. after being treated with 5% leaf extracts at 3 days after application.Bars (mean ± SE) with same letter(s) are not significantly different as determined by LSD test at P < 0.05.

Table 1 :
Plants and their parts used for evaluation of acaricidal activities against Luciaphorus sp.