First report of stem rot caused by Bipolaris cactivora on dragon fruit in Malaysia NurShanariah Balkis Binti Kamarudin

First report of stem rot caused by Bipolaris cactivora on dragon fruit in Malaysia
NurShanariah Balkis Binti Kamarudin, 134468, BGT 302: Basic Practical of Plant Pathology, School of Biological Sciences, University of Science Malaysia, Pulau Pinang, Malaysia.

INTRODUCTION
Dragon fruit (Hylocereus undatus) is one of the tropical fruits under cactus family, Cactaceae. It has a bright red skin fruit studded with green scales and white or red flesh (depends on the species) with tiny black seeds. In Malaysia, there are two species of the dragon fruit that commonly found which are Hylocereus undatus (white dragon fruit) and Hylocereus polyrhizus (red dragon fruit). Like other tropical fruits, it is believed that dragon fruit is rich in vitamins and minerals that have antioxidant properties to protect the body against free radicals that causing aging, cancer and disease. Dragon fruit also contain lycopene, a plant pigment in a red fleshed species that acts as an antioxidant to help reduce the risk of a heart attack ADDIN EN.CITE <EndNote><Cite><Author>Foundation</Author><Year>2016</Year><RecNum>6</RecNum><DisplayText>(Foundation 2016)</DisplayText><record><rec-number>6</rec-number><foreign-keys><key app=”EN” db-id=”e9ta9ze25e5ewzefdx25v2eqx52f5rp9xze5″ timestamp=”1542018080″>6</key></foreign-keys><ref-type name=”Online Multimedia”>48</ref-type><contributors><authors><author>American Culinary Federation Education Foundation</author></authors></contributors><titles><title>All about the dragon fruit</title></titles><pages>1</pages><keywords><keyword>Benefits of dragon fruit pdf</keyword></keywords><dates><year>2016</year><pub-dates><date>12 November 2016</date></pub-dates></dates><urls><related-urls><url>https://www.acfchefs.org/download/documents/ccf/nutrition/2016/201604_ingredient_dragon_fruit_flyer.pdf</url></related-urls></urls><custom1>2018</custom1><custom2>12 November</custom2><access-date>12 November 2018</access-date></record></Cite></EndNote>(Foundation 2016). Such a beneficial plant found suffering with a stem rot symptom has made us to diagnose the causal agent in order to control and prevent the disease from infecting on another plants.
Therefore, the objective of the experiment is to identify the causal agent causing the stem rot on the dragon fruit given based on a scientific method which is Koch Postulates by performing an isolation of the microorganisms from diseased plant, doing a morphological observation and pathogenicity test and applying an aseptic technique throughout the experiment.
Disease symptoms and pathogen description. The infected dragon fruit with a stem rot symptom is given in a laboratory at School of Biological Sciences, University of Science Malaysia on 4 October 2018. The infected dragon fruit suffers from a lesion on its stem. Below the lesion part, there is a yellow spot formed with a reddish margin. The infected region looks a little bit swollen but its surface is dry. When the infected region is dissected and observed under a dissecting microscope, there is a few of red spots found. There is no sign is observed.

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Fig. 1 A: Symptoms of stem rot on the dragon fruit, B: Image of the red spots under dissecting microscope of the infected region , C: Colonies of B. cactivora on PDA plates after 7 days of growth under intermittent ambient light at room temperature (23°C to 25°C) D: Hyphae of B. cactivora observed under 40 x lens of compound microscope from the six sub-cultured plates, E and F: Colonies of B. cactivora on Plate 3 and Plate 8 that are used for the pathogenicity test, G: Image of the inoculated plant in the positive control site under dissecting microscope after 7 days of the inoculation, H: : Image of the inoculated plant in the Culture 2 site under dissecting microscope after 7 days of the inoculation, I: Symptoms appear on the positive control site and Culture 2 site of the inoculated plant after 7 days, J: Spore of B. cactivora observed under 40 x lens of compound microscope, K: Conidiophore of B. cactivora observed under 40 x lens of compound microscope from the re-isolated plates, L: Colony colour of B. cactivora on the re-isolated culture.

MATERIALS AND METHOD
Isolation of microorganisms from diseased plant. The isolation of a pathogen from diseased plant is performed on 4 October 2018. Before the isolation process is started, an observation of the symptom and sign on the infected dragon fruit is done first. The symptoms on the infected plant is described, sketched and recorded on a log book. The symptoms are also recorded in the form of a photo. For the macroscopic view, the infected plant is observed by using a naked eyes and dissecting microscopes for a better view. For the microscopic view, the infected plant is observed by using a compound microscope. Then, the isolation process is carried out after enough information is collected from the observation. To start the isolation process, the surface of the plant is sterilized by using 70% of ethanol. The infected plant is cut at the margin of the infected and healthy tissue because it is where the pathogen is the most active. Then, the samples (eight samples) are soaked in 1% of sodium hypochlorite (NaOCl) for 3 minutes and rinsed in two changes of sterile water for I minute each by using a forceps. Aseptic technique is adopted throughout the isolation process. Sterile filter papers are used to remove the excess water from the samples after they are soaked. Next, the samples (eight samples) are transferred onto the two labelled PDA plates. Each PDA plates have four samples. After that, the plates are incubated under intermittent ambient light at room temperature (23°C to 25°C) for 3 to 5 days. On the fourth day of the incubation which is 7 October 2018, a sub-culture is done by transferring three different fungal colonies observed onto the new PDA plates. The new PDA plates are later used in the next experiment session.

Morphological Observation. The experiment is proceeded by observing the morphological character formed on the new sub-cultured PDA plates on 11 October 2018. The morphological observation is focused on the macroscopic characters and microscopic characters formed. Dissecting microscopes is used to observe macroscopic characters formed on the PDA plates. All the information about the colony colour and colony appearance observed on the PDA plates is recorded in a log book and in a form of a photo. Wet mounts is done in order to observe the microscopic characters formed under a compound microscope and all the information about the conidia and conidiogenous cells formed on the PDA plates is also recorded in a log book and in a form a photo. Two out of the six cultures are chosen for the pathogenicity test on the next experiment session. The chosen cultures are sub-cultured again onto the new PDA plates. Then, the new PDA plates are sealed with a Parafilm tape and labelled (Group, date and culture).

Pathogenicity test. In this experiment session, pathogenicity test is done which is on 18 October 2018. First step in performing pathogenicity test is to clean the healthy dragon fruit plant by spraying an alcohol on its surface. Next, a disposable syringe with needle is used to inoculate 0.5ml of the conidia suspension into the healthy plant. The healthy plant is inoculated in four sites on its stem surface. The first site is inoculated with a given culture and it is labelled as a positive control. The second site is inoculated with the Culture 1 (one of the two chosen cultures from the previous experiment session) and it is labelled as a Culture 1. Next, the third site is inoculated with Culture 2 (one of the two chosen cultures from the previous experiment session) and it is labelled as a Culture 2. The last site is inoculated with a sterile distilled water and it is labelled as a negative control. After the four sites are inoculated and labelled, the inoculated plant is placed in the School of Biological Science’s plant house for 7 days. The inoculated plant will be examined on 25 October 2018. To fulfil the Koch ‘s Postulates, the re-isolation of pathogen from inoculated dragon fruit is performed. The morphological characteristics of the re-isolated cultures are then compared with those of the original cultures.

RESULT
Morphological Observation. All of the pathogen colonies developed on the isolated PDA plates produces green moss colony in irregular form with a raised elevation (Fig. 1: C, Colonies of B. cactivora on PDA plates after 8 days of growth). Abundance of fluffy white mycelia also formed on the isolated PDA plates with undulate margin. When the pathogen colonies are observed under 40 x lens of a compound microscope, only hyphae are found and there are no conidia at all.

Pathogenicity test. Dragon fruit shows disease symptoms in 7 days after the inoculation. The symptoms only appear on the positive culture site, Culture 1, and Culture 2 site. The symptoms appear on Culture 2 site is more severe compared to Culture 1 but there is no symptoms appear on negative control site (Fig. 1, I: Symptoms appear on the positive control site, Culture site 1, and Culture 2 site of the inoculated plant after 7 days). It suffers from a lesion on its stem. Around the lesion part, there is a yellow spot formed with a reddish margin. The infected regions on the inoculated dragon fruit looks a little bit swollen and the surface is dry. When the infected region is dissected and observed under a dissecting microscope, there is a few of red spots found both in positive control site and Culture 2 site (Fig. 1, G: Image of the inoculated plant in the positive control site under dissecting microscope after 7 days of the inoculation, H: Image of the inoculated plant in the Culture 2 site under dissecting microscope after 7 days of the inoculation). The pathogen is re-isolated from the inoculated dragon fruit and cultured on PDA plates again. The re-isolated culture is found to produce a green moss colony in irregular form. It also forms fluffy white mycelia with undulate margin (Fig. 1, L: Colony colour of B. cactivora on the re-isolated culture. When the re-isolated culture is observed under 40 x lens of compound microscopes, there is a brownish spore with a septae like and conidiophores are found (Fig. 1, J: Spore of B. cactivora observed under 40 x lens of compound microscope, K: Conidiophore of B. cactivora observed under 40 x lens of compound microscope from the re-isolated culture).

DISCUSSION
Based on the morphological characteristics, the pathogen is similar to the fungus pathogen and identified as B. cactivora. Both isolated culture and re-isolated culture are morphogically identical. The isolated culture plate and re-isolated culture plate has produce green moss colony with irregular form. They also form fluffy white mycelia with undulate margin. When the isolated culture plate is observed under 40 x lens of compound microscope, there is no spore is found and only hyphae like is present. It is believed that the spore is not produced yet because usually it took 7 days to produce them. But the observation of the isolated culture plate is done on the fourth day. So, that is why there is no spore is found during the observation. For the re-isolated culture plate, there is a brownish spore with a septae like and conidiophores found. The re-isolated culture plate can produce the brownish spore because it is observed after 7 days of the re-isolation process. So, it is an enough duration growth for the B. cactivora to produce its spore. Besides, the infected dragon fruit is found to have similar symptoms appear on its stem as inoculated dragon fruit. Both have a lesion and form yellow spot with a reddish margin. When, they are being observed under dissecting microscope, both of them also found to have red spots. The similar symptoms appear on both of them because the causal agent of the disease is the same, B. cactivora which is later confirmed by satisfying Koch ‘s Postulates. The pathogen is re-isolated from the inoculated dragon fruit and cultured on PDA plates again. The pathogen colony forms on the re-isolated PDA plate is morphologically identical to the original pathogen isolated from the infected dragon fruit. Thus, all these characteristics fulfil Koch ‘s Postulates as the pathogen found is constantly associated with the diseased symptom, it is isolated from the infected dragon fruit and grown in the pure culture, healthy dragon fruit is inoculated with the pathogen from the infected dragon fruit and produce the same symptom as the original one and the pathogen is then re-isolated again. The morphological characteristics of the re-isolated pathogen found to be the same as the original pathogen causing the stem rot on dragon fruit. Therefore, it is proved that the causal agent causing the stem rot on dragon fruit is B. cactivora. Based on previous studies, the first report of stem rot disease caused by B. cactivora has been reported in Israel (Israel et al. 2011) whereas the B. cactivora also has been reported as a causal agent for the other disease such as fruit rot of dragon fruit in Japan (Taba et al. 2007), South Florida (Tarnowski et al. 2010), imported dragon fruit from Vietnam (Ho, 2012), and Thailand (Oeurn et al. 2015). In conclusion, by having this report it should help in recognizing the disease from symptoms on the plant and providing useful information for devising disease control.

REFERENCE
ADDIN EN.REFLIST Foundation, A. C. F. E. (2016). All about the dragon fruit: 1.

Ho, H. (2012). Bipolaris cactivora causing fruit rot of dragon fruit imported from Vietnam. Plant Pathology & Quarantine.

L. B. Tarnowski, T & J. Palmateer, A & Crane, Jonathan. (2010). First Report of Fruit Rot on Hylocereus undatus Caused by Bipolaris cactivora in South Florida. Plant Disease. 94. 1506-1506. 10.1094/PDIS-06-10-0406.

Oeurn, Samoul & Jitjak, Wuttiwat & Sanoamuang, Niwat. (2015). Fungi on Dragon Fruit in Loei Province, Thailand and the Ability of Bipolaris cactivora to Cause Post-harvest Fruit Rot. KKU Research Journal. 20. 405-418. 10.14456/kkurj.2015.34.

Taba, Satoshi & Miyahira, Nao & Nasu, Kanami & Takushi, Tetsuya & Moromizato, Zen-ichi. (2007). Fruit rot of Strawberry pear (pitaya) caused by Bipolaris cactivora. Journal of General Plant Pathology – J GEN PLANT PATHOL. 73. 374-376. 10.1007/s10327-007-0032-x.