# Effect of Crude Plant Extracts on Mushroom Mite,Luciaphorus sp. (Acari: Pygmephoridae)

Authors: Prapassorn Bussaman; Chirayu Sa-uth; Paweena Rattanasena; Angsumarn Chandrapatya
Journal: Psyche
 (2012)

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## Abstract

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.


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## Body

## 1. Introduction

Luciaphorussp. (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 [3–6]. 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, Luciaphorussp.


## 2. Materials and Methods

### 2.1. 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.Luciaphorussp. 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. squarrosulusspawn 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.


### 2.2. Preparation of Plant Extracts

Leaves and rhizomes of 23 plants were collected locally from Mahasarakham province in the Northeast of Thailand (Table1). Plant materials were cut into small pieces and dried in hot air oven at 45°C for 3 days.Table 1
Plants and their parts used for evaluation of acaricidal activities againstLuciaphorus sp.
Scientific name
Family
Common name
Parts
Boesenbergia  pandurata   (Roxb) Schltr.
Zingiberaceae
Fingerroot
Rhizome
Kaempferia parviflora Wall. Ex Baker
Zingiberaceae
Belamcanda chinensis
Rhizome
Kaempferia   pulchra (Ridl.) Ridl.
Zingiberaceae
Peacock ginger, resurrection lily
Rhizome
Zingiber zerumbet (L.) Smith.
Zingiberaceae
Wild ginger, Martinique ginger
Rhizome
Zingiber officinale Roscoe.
Zingiberaceae
Ginger
Rhizome
Zingiber montanum (Koenig) Link ex Dietr.
Zingiberaceae
Phlai, cassumunar
Rhizome
Alpinia galanga (L.) Swartz.
Zingiberaceae
Kha, galingale, galanga
Rhizome
Curcuma longa Linn.
Zingiberaceae
Turmeric
Rhizome
Curcuma xanthorrhiza Roxb.
Zingiberaceae
Curcuma
Rhizome
Cymbopogon citratus Stapf.
Gramineae
Takhrai, lemongrass
Leaf
Citrus hystrix DC.
Rutaceae
Leech lime
Leaf
Ocimum basilicum Linn.
Labiatae
Ho-ra-pa, sweet-basil, common basil
Leaf
Ocimum canum Linn.
Labiatae
Hairy basil
Leaf
Ocimum   sanctum Linn.
Malvaceae
Holy basil, sacred basil
Leaf
Moringa   oleifera Lam.
Moringaceae
Horse radish tree
Leaf
Annona   squamosa Linn.
Annonaceae
Sugar apple
Leaf
Psidium guajava Linn.
Myrtaceae
Guava
Leaf
Eucalyptus camaldulensis Dehnh.
Myrtaceae
Red river gum, Murray red gum, red gum
Leaf
Artocarpus heterophyllus Lam.
Moraceae
Jackfruit tree
Leaf
Piper sarmentosumRoxb. Ex Hunter.
Piperaceae
Cha-plu
Leaf
Murraya paniculata (L.) Jack.
Rutaceae
Orange jessamine, satin-wood,
Leaf
Melissa officinalis L.
Lamiaceae
Kitchen mint, marsh mint
Leaf
Cassia   siamea (Lam.) Irwin et Barnaby
Fabaceae
Kassod tree, siamese senna, Thai copperpod, siamese cassia
LeafThe 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).


### 2.3. 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 500μL 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.


### 2.4. Statistical Analysis

Data on the percentages of mite mortality due to application with plant extracts were arcsine-transformed 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 atP<0.05.


## 2.1. 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.Luciaphorussp. 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. squarrosulusspawn 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.


## 2.2. Preparation of Plant Extracts

Leaves and rhizomes of 23 plants were collected locally from Mahasarakham province in the Northeast of Thailand (Table1). Plant materials were cut into small pieces and dried in hot air oven at 45°C for 3 days.Table 1
Plants and their parts used for evaluation of acaricidal activities againstLuciaphorus sp.
Scientific name
Family
Common name
Parts
Boesenbergia  pandurata   (Roxb) Schltr.
Zingiberaceae
Fingerroot
Rhizome
Kaempferia parviflora Wall. Ex Baker
Zingiberaceae
Belamcanda chinensis
Rhizome
Kaempferia   pulchra (Ridl.) Ridl.
Zingiberaceae
Peacock ginger, resurrection lily
Rhizome
Zingiber zerumbet (L.) Smith.
Zingiberaceae
Wild ginger, Martinique ginger
Rhizome
Zingiber officinale Roscoe.
Zingiberaceae
Ginger
Rhizome
Zingiber montanum (Koenig) Link ex Dietr.
Zingiberaceae
Phlai, cassumunar
Rhizome
Alpinia galanga (L.) Swartz.
Zingiberaceae
Kha, galingale, galanga
Rhizome
Curcuma longa Linn.
Zingiberaceae
Turmeric
Rhizome
Curcuma xanthorrhiza Roxb.
Zingiberaceae
Curcuma
Rhizome
Cymbopogon citratus Stapf.
Gramineae
Takhrai, lemongrass
Leaf
Citrus hystrix DC.
Rutaceae
Leech lime
Leaf
Ocimum basilicum Linn.
Labiatae
Ho-ra-pa, sweet-basil, common basil
Leaf
Ocimum canum Linn.
Labiatae
Hairy basil
Leaf
Ocimum   sanctum Linn.
Malvaceae
Holy basil, sacred basil
Leaf
Moringa   oleifera Lam.
Moringaceae
Horse radish tree
Leaf
Annona   squamosa Linn.
Annonaceae
Sugar apple
Leaf
Psidium guajava Linn.
Myrtaceae
Guava
Leaf
Eucalyptus camaldulensis Dehnh.
Myrtaceae
Red river gum, Murray red gum, red gum
Leaf
Artocarpus heterophyllus Lam.
Moraceae
Jackfruit tree
Leaf
Piper sarmentosumRoxb. Ex Hunter.
Piperaceae
Cha-plu
Leaf
Murraya paniculata (L.) Jack.
Rutaceae
Orange jessamine, satin-wood,
Leaf
Melissa officinalis L.
Lamiaceae
Kitchen mint, marsh mint
Leaf
Cassia   siamea (Lam.) Irwin et Barnaby
Fabaceae
Kassod tree, siamese senna, Thai copperpod, siamese cassia
LeafThe 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).


## 2.3. 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 500μL 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.


## 2.4. Statistical Analysis

Data on the percentages of mite mortality due to application with plant extracts were arcsine-transformed 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 atP<0.05.


## 3. Results

### 3.1. Acaricidal Activities of Rhizome Extracts

In this study, all rhizome extracts were shown to have acaricidal activities againstLuciaphorussp., and the percentages of mite mortality increased progressively and reached the plateau after 3 days of applications (Figure 1). On day 3, the significantly high levels of mortality rates were caused by the rhizome extracts ofC. xanthorrhiza(100%), Z. montanum (100%), C. longa (98.89%),  and Z. zerumbet(97.78%), followed byK. parviflora (88.89%), Z. officinale(84.44%),  B. pandurata (80.00%),K. pulchra(72.22%),  andA. galanga (63.33%) (Figure 1). Interestingly, on day 1, K. parviflora, Z. officinale, C. longa, andC. xanthorrhizaextracts resulted in mortality rates at over 70% which were significantly higher than the other treatments (data not shown). However, on day 2, mite mortality rates in almost all treatments were over 70% with the exception of A. galanga(56.67%) and K. pulchra (67.78%) (data not shown).Figure 1
The mortality rates of adult femaleLuciaphorussp. 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.


### 3.2. Acaricidal Activity of Leaf Extracts

The levels of mite mortality after applications with leaf extracts also reached maximum on day 3 (Figure2). On day 3, the leaf extracts ofO. sanctumand M. officinalisresulted 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. sanctumandM. 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. sanctumandM. officinalis still resulted in mite mortality at the levels significantly higher than the rest, accounting for 97.78% and 94.44%, respectively (data not shown).Figure 2
The mortality rates of adult femaleLuciaphorussp.  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.


## 3.1. Acaricidal Activities of Rhizome Extracts

In this study, all rhizome extracts were shown to have acaricidal activities againstLuciaphorussp., and the percentages of mite mortality increased progressively and reached the plateau after 3 days of applications (Figure 1). On day 3, the significantly high levels of mortality rates were caused by the rhizome extracts ofC. xanthorrhiza(100%), Z. montanum (100%), C. longa (98.89%),  and Z. zerumbet(97.78%), followed byK. parviflora (88.89%), Z. officinale(84.44%),  B. pandurata (80.00%),K. pulchra(72.22%),  andA. galanga (63.33%) (Figure 1). Interestingly, on day 1, K. parviflora, Z. officinale, C. longa, andC. xanthorrhizaextracts resulted in mortality rates at over 70% which were significantly higher than the other treatments (data not shown). However, on day 2, mite mortality rates in almost all treatments were over 70% with the exception of A. galanga(56.67%) and K. pulchra (67.78%) (data not shown).Figure 1
The mortality rates of adult femaleLuciaphorussp. 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.


## 3.2. Acaricidal Activity of Leaf Extracts

The levels of mite mortality after applications with leaf extracts also reached maximum on day 3 (Figure2). On day 3, the leaf extracts ofO. sanctumand M. officinalisresulted 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. sanctumandM. 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. sanctumandM. officinalis still resulted in mite mortality at the levels significantly higher than the rest, accounting for 97.78% and 94.44%, respectively (data not shown).Figure 2
The mortality rates of adult femaleLuciaphorussp.  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.


## 4. Discussion

Several plants have been found to contain bioactive compounds with a variety of biological actions against insects and mites, including repellent, antifeedant, anti-ovipositional, 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 urticaeKoch) [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 LD50 of 246 ppm [12]. Moreover, Aslan et al. [13] reported that essential oil vapours from Satureja hortensisLinn., Ocimum basilicumLinn, and Thymus vulgarisLinn. had potential against T. urticae, but the essential oil obtained from S. hortensiswas 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. cinnabarinusand T. viennensiscould be quickly induced by the petroleum ether extract of Juglans regiaLinn.,resulting in mortality rates at 81.58 and 78.58%, respectively, within 24 h [7].Furthermore, the complete 100% mortality ofT. cinnabarinus was found to be induced by the essential oils of Cuminum cyminumLinn., Pimpinella anisum Linn., and Origanum syriacum var. bevaii (Holmes) as fumigants in greenhouse experiments [15]. This complete mortality could also be produced by using the acetone parallel extract of Artemisia annua Linn. leaves collected in July [16]. In addition, Zhang et al. [17] reported that benzene extracts derived from C. longaLinn. had LC50 againstT. cinnabarinusat 99.3 ppm after 72 h. The high mortality rates of T. cinnabarinus could be induced by methanol extracts of Gliricidia sepium (Jacq) Kunth ex Steud. (100%) andLippia origanoides Kunth (96.6%) when used at the concentration of 20% [18]. Additionally, Sertkaya et al. [8] evaluated the efficacy of essential oils derived from medicinal plants against T. cinnabarinus and showed that thyme (Thymbra spicata Linn. subsp. spicata), oregano (Origanum onites Linn.), mint (Mentha spicata Linn.), and lavender (Lavandula stoechas Linn. subsp. stoechas) essential oils had LC50 values of 0.53, 0.69, 1.83, and 2.92 ppm, respectively.Moreover, the acetone extract of Aloe vera Linn. leaves was shown to have acaricidal activity against female T. cinnabarinus at 3 days after treatment with LC50 value of 90 ppm [6].Other insect pests were also found to be inhibited by plant extracts. According to the results of Liu et al. [19], the ethanol extracts of Eupatorium adenophorum Spreng. (0.1% w/v) could cause mortality of citrus red mite (Panonychus citri(McGregor)) at 71.10 and 73.53% after 12 and 24 h, respectively. Also, the activities against P. citri of the ethanol extracts derived from Boenninghausenia sessilicarpa H. Lev., Laggera pterodonta (DC.) Benth., Humulus scandens (Lour) Merr., and Rabdosiawere reported with LC50 values of 0.9241, 0.9827, 0.9905, and 1.0196 mg/mL, respectively [20]. In addition, applications with aqueous extracts of Acorus calamus Linn., Xanthium strumarium Linn., Polygonum hydropiper Linn., and Clerodendron infortunatum (Gaertn.) could lead to more than 50% mortality of Oligonychus coffeae (Nietner) [21]. Moreover, 3% methanolic extracts of Ocimum tenuiflorumLinn. and Cassia alata Linn. exhibited acaricidal activities against Tetranychus neocaledonicus Andre. and resulted in the mortality at 93.3 and 97.0%, respectively [22]. On the other hand, 3% aqueous extracts of C. alata and O. tenuiflorum could lead to mortality of T. neocaledonicus at 75% and 82.2%, respectively, after exposure for 3 days. In addition, the volatile oils of Citrus reticulata Blanco. and C. longa Linn. could cause mortality of Sitophilus oryzae Linn. as high as 100 and 90%, respectively [23]. The essential oils of Ocimum basilicum Linn., Coriandrum sativum Linn., Eucalyptus globulus Labill, Mentha piperita Linn. and Satureja hortensis Linn. were toxic against poultry red mite (Dermanyssus gallinae (De Geer)), and, when using the in vitro direct contact method, these essential oils at the dose of 0.6 mg/cm could result in mortality rates over 80% after 24 h of contract [24]. Furthermore, Eucalyptus citriodora Hookextract was found to be effective against D. gallinae, resulting in 85% mortality over a 24 h exposure period in contact toxicity tests [25].In this study, the rhizome extracts ofC. 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 Luciaphorussp. 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 perniciosusRack., and the median effective concentration (EC50) was 18.15 and 19.66 ppm, respectively [26]. In addition, the essential oils of Litsea cubebaPers. were effective against L. perniciosusby contact and fumigation methods with LD50 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 ofC. xanthorrhizaand Z. montanumand the leaves ofO. sanctumand M. officinalis for controlling Luciaphorus mites. The effective concentration and mode of action of these plant extracts againstLuciaphorus sp. remain to be determined for the future development of highly potent products to be used in the real settings.



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*Source: 150958-2012-07-09.xml*