First Report of Necrotic Spot Disease Caused by Cactus virus X on Dragon Fruit ( Hylocereus spp.) in Peninsular Malaysia

This study was conducted to detect the status of virus causing necrotic spot disease on dragon fruit and to recognize its geographical distribution in Peninsular Malaysia. Fifty posts of crops were randomly sampled from dragon fruit orchards. The symptoms were characterized and the pathogen was observed under transmission electron microscopy (TEM). Disease incidence and severity were plotted, while the disease occurrence was statistically analyzed under Duncan’s Multiple Range Test (DMRT) of General Linear Modeling (GLM) procedure and Pearson correlation test. The disease was initiated by necrotic small mottle or spot on young shoot which then turned to orange. Those symptoms were also found on mature stems and fruits. They could quickly expand on unexposed symptomatic parts in orchards. The maximum incidence and severity of disease were recorded in Durian Tunggal (Malacca), i.e. 98 and 52%, respectively. TEM technique viewed the spindle-shaped inclusion bodies of virus within symptomatic stems at 4000 and 20,000× magnification which were suspected as Cactus virus X (CVX), a potexvirus with filamentous and rod shape and 515−520 nm in size. Statistically, disease occurrence in Malacca was significantly higher than those in other states, while the minimum was found in Terengganu. The strong regression of disease incidence and severity was shown by R value= 0.9484. There were no significant correlations of disease occurrence either with weather or cultural factors. Further study on the transmission of virus within the crops in the field is important to be carried out in order to monitor the spread of disease and to develop the integrated disease management strategies on dragon fruit in Peninsular Malaysia.


INTRODUCTION
Dragon fruit (Hylocereus spp.) is a climbing cacti species bearing the fruit characterized by dragon-like scales covering its skin (Mizrahi et al., 2002). It is popular fruit in South America and has been consumed by the general population since the pre-Columbian times (Crane & Balerdi, 2005). French brought it into Vietnam about 100 years ago as ornamental crop for the king (Luders & McMahon, 2006).
The commercial cultivation of dragon fruit in Malaysia was initiated in Kluang (Johore), Kuala Pilah (Negeri Sembilan) and Sitiawan (Perak) at the beginning of 1999. However, the Golden Hope Company, which is located at Sungai Wangi Estate (Perak), had introduced and cultivated this crop in a large scale plantation two decades ago (Halimi & Satar, 2007). According to the statistics reported on 2006, Malaysia planted around 962.3 ha of dragon fruit with the total production about 2,534.2 ton (production value around US$3.5 millions) (Cheah & Zulkarnain, 2008).
However, there is no scientific report regarding on the occurrence and distribution of this pathogen in Peninsular Malaysia. Therefore, this study was carried out to detect the presence of plant pathogenic virus causing disease on dragon fruit and to recognize its geographical distribution on dragon fruit-growing areas in Peninsular Malaysia.

Surveys of Dragon Fruit-Growing Areas
Surveys were conducted in dragon fruit-growing areas in Peninsular Malaysia since December 2007 until August 2008. At least two dragon fruit orchards from each sampling state were surveyed. Data of disease occurrence and cultural (age of crops and acreage of surveyed farms) were recorded. Longitude and latitude data were documented using GPS device (SILVA Multinav-Version 2.01) and then mapped with Mapinfo Software (Troy, New York; USA). Weather data i.e., relative humidity (RH), temperature and wind velocity were obtained from Malaysian Meteorological Department (period of 1998-2008).

Assessment of Disease
Fifty posts of dragon fruit plants were sampled using pseudo-random method with specifying an approximate W-shaped path from each surveyed plantation with at least about 0.4 ha in acreage. Each plot was generally occupied by 4 cuttings. Disease incidence (DI) was calculated by the following equation (Cooke, 2006):

RESULTS AND DISCUSSION
Cactus virus X (CVX) is considered as one of important plant pathologic virus on cacti species (Zimmermann & Granata, 2002). Early detection of CVX on dragon fruit (Hylocereus spp.) in Peninsular Malaysia could be the basic information in developing the appropriate strategies to prevent the spread of disease, particularly in investigating the virus transmission within crops in the field and in selecting the resistant dragon fruit varieties.

Surveys of Dragon Fruit-Growing Areas
The present study recorded that the red-fleshed species was planted nationwide in Peninsular Malaysia, followed by white-fleshed and yellow species, with age of crops 0.25−6 years and 0.4−10.11 ha in acreage and the altitude of 3.35−146.91 m above sea level (asl) ( Table 1). The 43 surveyed orchards and the distribution of disease could be mapped as shown by Figure 1. The disease was widely distributed in Peninsular Malaysia and could quickly expand on unexposed infected plant parts in dragon fruit orchards. The 11year of average weather data in Peninsular Malaysia affecting the establishment of current surveyed disease occurrences was also given in Figure 2.

Assessment of Disease
This disease initially appeared as necrotic small mottle or spot on base of young shoots and unripe fruits which later turned to orange. The secondary found pathogens such as Alternaria sp., Botryosphaeria sp., Colletotrichum gloeosporioides, Curvularia sp., Fusarium sp., Monilinia sp., Pestalotiopsis sp., Phytophtora sp., and Thielaviopsis sp. might cause the rot of infected tissues. Those symptoms were also found on some mature stems and ripe fruits ( Figure 3). The highest disease occurrence was recorded in Durian Tunggal (Malacca), with DI and DS about 98 and 52%, respectively (Table 1). It was presupposed that the most severe disease in this orchard likely due to unwell-sanitized farm. The farmer did not prune the damaged stems and ignored debris of diseased stems around the farm ( Figure 4).
The lowest disease prevalence was found in Mata Ayer (Kedah), while there were some zero incidences of disease in certain surveyed areas (Table 1). We found that the location of those areas were far away from infected orchards. Several farms were possibly not contaminated by this disease as they were wellsanitized by pruning, burning and then burying the in-

Detection of Plant Pathogenic Virus
Suspected pathogenic virus was characterized by observing size and shape under Transmission Electron Microscopy (TEM) (LEO912AB, EFTEM) following procedure provided by Institute of Biological Science, University Putra Malaysia. The tissues were cut into 1 mm 3 slices and then put into fixative solution (4% of Glutaraldehyde) for 48 h at 4 o C. Afterwards, the slices were washed with 0.1 M Sodium Cacodylate Buffer for three times in 30 min each and post-fixed with 1% Osmium Tetroxide solution for 2 h at 4 o C. Again, the slices were washed with 0.1 M Sodium Cacodylate Buffer for three times in 30 min each. They were consecutively involved in serial dehydration within 35, 50, 75 and 95% acetone for 60 min each, as well as in 100% acetone during 120 min for three times.
Hereinafter, the slices were infiltrated into mixture of acetone and resin with the ratio of 1:1 and 1:3 for one day and one night, respectively, and 100% resin overnight and 2 h, consecutively. The samples were then placed into resin-filled up beam capsules and polymerized in oven at 60 o C for 24-48 h.
Thick sectioning procedures were started with the cutting of samples into 1 μm sections using glass knife and ultramicrotome. They were then placed onto glass slide and stained with toluidine blue; the slides were dried on hot plate, while the stain was washed; the slides were observed under light microscope.
Meanwhile thin sectioning was initiated by the selection of interested area on samples. They were then cut for ultrathin section; the silver or golden sections were selected and picked up with a grid and dried using filter paper. The sections were consecutively stained and washed with uranyl acetate for 10 min and filtered alcohol, followed by lead-staining for 10 min and rinsing with double-distilled water. Finally, the samples were viewed under TEM.

Statistical Analysis
Test of normality for disease occurrence data (DI and DS) was previously employed (Cardoso et al., 2004).       fected plants out of the orchards. Le Bellec et al. (2006) recommended pruning all the injured and entangled stems at the first year after planting. Some orchards were regularly treated with effective microbe and application of insecticide, such as in Sepang (Selangor) and Jerantut (Pahang), respectively. Moreover, this disease was encountered in all sampling states with the range of disease frequency around 40−100% (Table 1). This indicated that necrotic spot disease has infected at least one dragon fruit orchard in each surveyed state.

Detection of Plant Pathogenic Virus
Viral infection was detected on young stem with as necrotic small mottle or spot symptoms. TEM technique viewed the spindle-shaped inclusion bodies of suspected virus on infected stems at 4000 and 20,000X magnification ( Figure 5). The inclusion bodies with similar shape have been observed in infected barrel cactus [Ferrocactus acanthodes (Lemaire) et al., 1978) and whitefleshed species of dragon fruit (Fudl-Allah et al., 1983) in California (USA).
Such inclusion bodies were suspected as aggregation of Cactus virus X, a filamentous rod potexvirus with 515−520 nm in size, which correspondingly caused distorted areoles, malformed spines, necrotic (Attanthom et al., 1978) stunted and systemic mottle symptoms (Fudl-Allah et al., 1983). That virus was also reported infecting same species in Taiwan with such symptoms on the infected white-fleshed dragon fruit (Liou et al., 2001;Liao et al., 2003) and on both white-fleshed and yellow species of dragon fruit in Okinawa Prefecture, Japan (Keiko & Miki, 2001). Its complete nucleotide sequence and genome organization have been determined as well (Liou et al., 2004). Attanthom et al. (1978) characterized that this virus was transmissible by plant sap inoculation and grafting.

Statistical Analysis
The relationship of DI and DS for this surveyed disease was indicated by value of R 2 = 0.8995 with coefficient correlation, R = 0.9484 ( Figure 6). This value clarified that the incidence of disease could strongly affect the increase of disease severity level. Seem (1984) noted that such relationship could create an epidemiologically significant concept. It meant that the diseases occurred more severe and endemic unless control measures were immediately applied. Zimmermann and Granata (2002) explained once the wounded part of cacti plants was infected, disease development in the tissue was very rapid as the biochemical characteristics of their juicy cells were appropriate for the growth conditions of various biotic agents.
Statistical analysis showed that necrotic spot disease took place maximum in Malacca, with mean of incidence and severity level around 72.00% and 30.00%, respectively. In contrast, the minimum occurrence of disease was found on dragon fruit crops which were planted far away from infected orchards in Terengganu, i.e. around 13.60% and 4.00% for mean of DI and DS, respectively (Table 1).
Neither with climatic nor cultural factors had significant the Pearson coefficient ( Table 2). The incidence and severity of disease weakly correlated with those factors indicated by both small negative and positive of r values. Higher coefficient correlations were shown by correlation of disease prevalence with age of crops and acreage of orchard. These results explained that disease frequently occurred and more severe on older plants as well as on larger plantation.

CONCLUSION
Necrotic spot disease has been widely detected in dragon fruit-growing areas in Peninsular Malaysia with necrotic small mottle or spot which later turned orange and rotted symptomps. The spindle-shaped inclusion bodies of virus, which was then suspected as Cactus virus X, have been observed with TEM technique. The most severe disease significantly occurred in Malacca, while the less occurrence was recorded in Terengganu. No considerable correlation  was found between disease prevalence either with weather or cultural factors. Further study on the transmission of virus and disease-resistant variety of dragon fruit is important to be carried out in order to monitor the spread of disease and to develop the integrated disease management strategies on dragon fruit in Peninsular Malaysia.