Incidence of pests and viral disease on pepino (Solanum muricatum Ait.) in Kanagawa Prefecture, Japan

Abstract Background The solanaceous fruit crop pepino (Solanum muricatum Ait.), originating in the Andes, is grown commercially in South American countries and New Zealand. In these areas, pests and diseases of pepino have been identified well; however, to date, these have seldom been investigated in detail in Japan. Herein, we attempt to reconstruct an agricultural production system for commercial pepino crops in Japan, and evaluate the incidence of pests and viral diseases on pepino. The findings of this study will facilitate in developing a better crop system for the commercial cultivation of healthy pepino fruits. New information A total of 11 species, comprising nine insects and two mites, were recognized as pests of pepino plants in our experimental fields in Kanagawa Prefecture, central Honshu, Japan. Of these pest species, the two-spotted spider mite Tetranychus urticae Koch, 1836 and the cotton aphid Aphis gossypii Glover, 1877, were remarkably abundant than the other pest species. Eventually, 13 species, including two previously recorded, are currently recognized as the pests of pepino in Japan. With regard to viruses, we tested two species Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV), as well as three genera Carlavirus, Potexvirus, and Potyvirus. No virus was detected in symptomatic pepino leaves collected in our experimental fields. This is a first report on the identification of pests on pepino plants in Kanagawa Prefecture, Japan and elucidates the relationship between currently occurring pests of pepino plants and potential viral pathogens that they can transmit.


Introduction
Pepino, the Spanish name for sweet cucumber, (Solanum muricatum Ait.), is a solanaceous plant cultivated as a fruit crop. It originated in the Andes, became popular in several countries and regions of South America (Heiser 1964), and then it was introduced to Central America and New Zealand. In Japan, cultivation of pepino began in 1984 based on pepino fruits imported from New Zealand in 1983 (Sakata 2011). Since then, pepino caused a major boom and its cultivation rapidly spread throughout Japan within a few years. However, the production gradually declined prior to 1990 due to the low soluble solids content (Brix) in the Japanese pepino fruits (less than 8°Brix) (Sakata 2011). Currently, farmers in Japan do not grow pepino, except for people with gardening as a hobby, who cultivate pepino.
In 2016, our research team began a project for regional development "Launching of Nodaibranded Pepino Crop" conducted by Faculty of Agriculture, Tokyo University of Agriculture (TUA; Nodai is a Japanese abbreviated name of the university). The main purpose of this project was to produce high quality and flavorsome pepino fruits with sufficient soluble solids content. As a recent achievement of this project, Takahata (2017) succeeded in increasing the soluble solids content of pepino fruits by using a washer ring (metal washer) at the bottom of the stem. This technique strongly contributes to improving the quality and flavor of pepino fruits.
To date, at least 24 species of insect and mite pests on pepino (Larraín 2002) and one virus infected to pepino (Jones et al. 1980) have been recorded in the native range of pepino, the Andes. In contrast, in regions where pepino was introduced other than Japan, such as New Zealand, China and Turkey, there are only a few records on the pests (two species: Galbreath andClearwater 1983, Akyazi 2012) and viruses (two species: Thomas et al. 1980, Abouelnasr et al. 2014 in the academic literature. In Japan, little has been known in detail about the pests and viruses of pepino, except that inadequately identified pests such as spider mites and aphids damage to pepino.
It is important to establish solid pest control in its commercial cultivation to produce high quality and stable pepinos. Unfortunately, however, no pesticides applicable to pepino plants have been registered in Japan; this could be attributed to the few detailed studies on pests and diseases of pepino. Therefore, our research team has tried to comprehensively elucidate the pests and viral diseases of pepino in this project in order to contribute to the accumulation of basic knowledge toward the establishment of its pest control. This paper documents the results of our field survey on pests and diseases of pepino in Kanagawa Prefecture, central Japan.

Study sites
This study was conducted at the Atsugi Campus (35.432N 139.346E; at altitudes between 25 and 62 meters above sea level) of Tokyo University of Agriculture (TUA), Atsugi City, Kanagawa Prefecture, Japan, which is surrounded by residential quarters and a woody and grassy park (Fig. 1). The total site area of the campus is approximately 17.3 ha, within which several greenhouses and open fields for experimental use are present. The study site is located in a warm-temperate climate zone and has an annual mean temperature of 15.3 °C and annual mean precipitation of 1,729.9 mm (Japan Meteorological Agency 2017). In the campus, three survey plots were set ( Fig. 1); one of these was an open field (approximately 70 m ), where 40 pepino plants were cultivated (Plot A) (Fig. 2); another was a greenhouse (approximately 53 m ), in which 60 pot pepino plants were grown (Plot B) (Fig. 3); and the other was also a greenhouse (approximately 90 m ), in which approximately 400 pot pepino plants were grown (Plot C) (Fig. 4). In these plots, acaricides were applied approximately every two weeks in order to prevent pepino plants from withering due to mites, except for 20 pepino plants in Plot A; when the density of mites became high, appropriate chemicals were sprayed. All the plots were located within a radius of 250 m.    Incidence of pests and viral disease on pepino (Solanum muricatum Ait.) ...

Sampling methods for insects and mites
All specimens were collected by beating the leaves and branches of pepino plants after observation in field. A total of 34 collections were performed in the three plots from August 30, 2016 to January 21, 2017, for a maximum of 3 h/day in the daytime. The collected insects were killed immediately after capture, using ethyl acetate; aphids, lepidopteran larvae, and mites were fixed in plastic bottles filled with 70-80% ethanol. All specimens were prepared as dry mounted, slide mounded, or ethanol preserved for morphological examination.

Identification methods for insects and mites
Identification of insect and mite specimens was performed using stereoscopic microscopes (Olympus SZ60 and Olympus SZX16, Tokyo, Japan) and optical microscopes (Olympus BH-2 and Olympus BX41, Tokyo, Japan) by TI and YY according to the following literature: Ehara andGotoh (2009), Furukawa (2005), Harada and Takizawa (2012), Iwasaki et al. (2000), Kawai (1980), Masumoto and Okajima (2013), Matsumoto (2008), Moritsu (1983), Tanaka and Uesato (2012), Umeya and Okada (2003), Yasunaga et al. (2001), Yasunaga et al. (2015), along with the original descriptions and/or redescriptions of corresponding species if necessary. Collected specimens were regarded as pests in case these were directly damaging insects or mites on pepino plants, were known as pests of pepino in the native range and introduced regions of pepino other than Japan, or were known as pests of major solanaceous crops such as tomato, eggplant, green pepper, and potato, in Japan, with a reference to The Japanese Society of Applied Entomology and Zoology (2006). All examined specimens are preserved in the Insect Collection (IC) at the Laboratory of Entomology, TUA (LETUA).

Observation of virus-like diseases and virus detection
We surveyed whether pepino plants showed symptoms of virus infection such as mosaic, mottle, necrosis, or chlorosis. The symptomatic leaves were collected and used for virus detection as follows: Total RNA was extracted from the samples using Trizol reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's instructions. Total RNA was used as a template for first-strand cDNA synthesis by ReverTra Ace -α-kit (TOYOBO Co., Ltd., Osaka, Japan) followed by DNA amplification using TaKaRa Ex Taq PCR buffer (Takara Bio Inc., Otsu, Japan) with genus-specific or species-specific primers (

Data resources
In this study, a total of 498 specimens of insects and mites were collected from pepino plants on the three studied plots. Of these specimens, 459 individuals belonging to 11 species were recognized as pests of pepino. They consisted of nine insect species belonging to eight families of five orders and two mite species in two families of one order (  (Banks, 1904), were found only in greenhouses (Plots B and C); the former two are well known as glasshouse pests in Japan. No pest species were common in all the three plots. On an empirical basis, through our survey in the plots, two pest species, the two-spotted spider mite Tetranychus urticae Koch, 1836 and the cotton aphid Aphis gossypii Glover, 1877, were much more abundant on pepino plants than the other pest species recognized, with several hundreds of these two species on each pepino plant. Regarding virus-like diseases in our research fields, pepino leaves showing necrosis were rarely found in the greenhouse (Fig. 5). However, upper leaves showing mottle symptoms with deformation were remarkably observed only in the acaricide-untreated pepinos in the open field (Plot A) during September to October (Fig. 6). Those symptomatic leaves were tested for the detection of Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV), as well as the genera Carlavirus, Potexvirus, and Potyvirus. All tested pepino leaves showed no infection of the above-mentioned viruses (data not shown).    Furusato (1986) Arachnida Acari unspecified mites - Furusato (1984), Kita (1986), Odagiri et al. (1986) Arachnida Acari Tetranychidae spider mites - Takahashi (1985), Takagi (1985), Ozawa (1986), Takahashi (1986) Arachnida Acari Tetranychidae -Tetranychus urticae Koch, 1836Ozawa (1986 Currently, 25 species of insects and mites have been reported as pests of pepino plants worldwide (Table 4) (Galbreath and Clearwater 1983, Larraín 2002, Akyazi 2012. Among these pests, 16 species are distributed in Japan, but only three species, Trichoplusia ni, Polyphagotarsonemus latus, and Tetranychus urticae, were evaluated as pests of pepino in the present study. This indicates that the remaining 13 species are very likely to be Table 3.

Checklist of insect and mite pests of pepino in Kanagawa, Japan
Insect and mite pests of pepino plants previously recorded in Japan.
potential pests on pepino in Japan. Therefore, at least 26 insect and mite species, including the 13 currently known and the 13 potential ones in Japan, will be recognized as pests of pepino in the near future if the cultivation of pepinos spreads throughout the Japanese Archipelago.

Class
Order Family Species Country recorded as a pest dispersion throughout 38 prefectures by 2014 since the occurrence of TYLCV on tomato was first reported in Japan (Kato et al. 1998, Matsuura and Hoshino 2008, Ohnishi et al. 2016. However, there is no report of TYLCV incidence on pepino plants to date. Pepino can be regarded as a potential host for TYLCV through host adaptation or mutant as long as TYLCV-acquired vectors are present. Therefore, continuous monitoring of the distribution of both TYLCV and its vector is required. Some other species of insects and mites found on pepino plants in our research fields are also regarded as virus vectors ( Potato virus X (PVX)*2 Table 5.

Insects or mites of pepino plants involved in virus transmission
We found virus-like symptoms, showing mottle and deformation on pepino leaves. However, none of the tested viruses were detected in any plant. Our internet-based image searching results showed that the symptoms were similar to those on pepper or chili plants by Polyphagotarsonemus latus. Grinberg et al. (2005) described that young leaves were usually affected by P. latus and consequently showed distortion and leaf-curl downwards. This suggests that our symptomatic leaves might be caused by P. latus. Further research is required to reveal any effect of P. latus on pepino.
No related virus was detected in the present study, whereas two virus species had been detected from pepino plants in Japan: Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV) inducing chlorotic ring spots and mosaic symptoms on pepino plants, respectively (Honda et al. 1986). Moreover, Pepino mosaic virus (PepMV) was first isolated from pepinos showing yellow mosaic symptoms in Peru in 1974 (Jones et al. 1980). Currently, PepMV has become a major pathogen of tomato plants worldwide and is one of the quarantine pathogens strictly prohibited from entry into Japan. Pepino latent virus (PepLV; later reclassified as Potato virus S (PVS)) was detected from pepino cuttings in New Zealand, even though pepino plants with no symptoms had been imported from Chile (Thomas et al. 1980). With the increase in pepino production in China, Potato virus H (PVH) infected pepino, with no obvious symptoms (Abouelnasr et al. 2014) ( Table 6). Pests and pathogens in many crops cause tremendous losses both in terms of quantity and quality. Monitoring, detection, and identification of pests and pathogens prior to the introduction of a new crop or during production are important to efficiently regulate their future damage. Our findings will aid in understanding the incidence of pests and viral diseases on pepino plants and developing better crop production systems to combat pests and diseases.  Viruses infecting pepino plants naturally around the world.