Detection and Development of Infestation Rate of Aphelenchoides besseyi on Various Rice Seed Varieties

Rice is an important crop in Indonesia. In 2014, 14.30 million hectares produced 71.33 million tons of dry milled (Sembiring, 2015 citHabibi et al., 2016). In rice cultivation, Aphelenchoides besseyi come as an obstruction that carried and lives as ectoparasites on seeds and causes white tip (Tulek et al., 2015). This nematode was originally classified as OPTK A2 and has changed its status to become regular pest (regulated pest) since 2018 (Mentan RI, 2018). A. besseyi morphology is seen from its slim and long body shape and has stomato stylet type. The size of the median bulb is three-quarter of body width. There is an overlapping between the esophagus and the intestine of A. besseyi. Another characteristic of A. besseyi is that there is a mucro at the tip of the tail (Efendi, 2016). An understanding of the characteristics morphological and biology of plant parasitic nematodes is important step in the detection and management of plant parasitic nematodes. Rice seeds infested by A. besseyi are the main inoculum in its deployment in the world (Pashi et al., 2017). While in the grain (between the grain skin and rice seeds), A. besseyi is in dormant condition until three years after harvest. After the rice seeds were spread and germinate, A. besseyi will reactivate and move towards the growing point on pseudostems or leaves and eat plant tissue ectoparasitically (Azizah, 2017). Seeds infested by A. besseyi form different shapes and bear black spots on the skin. Not only on seeds, symptoms also found in plants which can be seen at the beginning of growth that shows white tip in the newly emerged leaves. The tips of the leaves dry, curl and twist, while the other leaves remain normal. The incidence of white tip disease tends to elevate along with the increase of the initial nematode population per gram of seeds and could lead to yield loss (Tulek et al., 2015). Yield loss due to A. besseyi has been widely studied. On infested land, yield losses generally range from 10–30%. On land where all crops were attacked, yield loss reaches 70% for susceptible cultivars and 20% for resistant cultivars (Prot, 1992). The amount of yield loss varies depending on cultivar, plant age, temperature, farming method, and other factors (Tulek & Cobanoglu, 2010). ABSTRACT


INTRODUCTION
Rice is an important crop in Indonesia. In 2014, 14.30 million hectares produced 71.33 million tons of dry milled (Sembiring, 2015cit Habibi et al., 2016. In rice cultivation, Aphelenchoides besseyi come as an obstruction that carried and lives as ectoparasites on seeds and causes white tip (Tulek et al., 2015). This nematode was originally classified as OPTK A2 and has changed its status to become regular pest (regulated pest) since 2018 (Mentan RI, 2018).
A. besseyi morphology is seen from its slim and long body shape and has stomato stylet type. The size of the median bulb is three-quarter of body width. There is an overlapping between the esophagus and the intestine of A. besseyi. Another characteristic of A. besseyi is that there is a mucro at the tip of the tail (Efendi, 2016). An understanding of the characteristics morphological and biology of plant parasitic nematodes is important step in the detection and management of plant parasitic nematodes.
Rice seeds infested by A. besseyi are the main inoculum in its deployment in the world (Pashi et al., 2017). While in the grain (between the grain skin and rice seeds), A. besseyi is in dormant condition until three years after harvest. After the rice seeds were spread and germinate, A. besseyi will reactivate and move towards the growing point on pseudostems or leaves and eat plant tissue ectoparasitically (Azizah, 2017).
Seeds infested by A. besseyi form different shapes and bear black spots on the skin. Not only on seeds, symptoms also found in plants which can be seen at the beginning of growth that shows white tip in the newly emerged leaves. The tips of the leaves dry, curl and twist, while the other leaves remain normal. The incidence of white tip disease tends to elevate along with the increase of the initial nematode population per gram of seeds and could lead to yield loss (Tulek et al., 2015).
Yield loss due to A. besseyi has been widely studied. On infested land, yield losses generally range from 10-30%. On land where all crops were attacked, yield loss reaches 70% for susceptible cultivars and 20% for resistant cultivars (Prot, 1992). The amount of yield loss varies depending on cultivar, plant age, temperature, farming method, and other factors (Tulek & Cobanoglu, 2010).

Research on the detection and development of
A. besseyi attack rates on various rice seed varieties was carried out to obtain the certainty about cultivar and the development of A. besseyi attack rates every week.

Sampling
Rice seeds were taken randomly from one distributor in Karanganom, Polanharjo, and North Klaten District, which are thought to be the centers of market seeds by the farmers. Samples were taken 4-8 varieties of rice seeds randomly from each distributors, with 250 grams per each variety.

Detection of the Existence of Aphelenchoides besseyi in Seeds
Seeds that have been obtained from the field were then detected to observe the presence or absence of A. besseyi. The number of observed samples was 100 seeds with A. besseyi symptoms. Detection of the presence of nematodes in seeds was done by Hoshino and Togashi method (1999). Seeds were cut lengthwise and put in a 1 ml tip pipette (7 cm long, 1 mm in diameter and bottom 7.4 mm). A pipette tip filled with 6 ml of water was incubated at 25°C for 24 hours. Next, the water in the vial and tip pipette (nematode suspension) were transferred in a counting dish to count the number of nematodes and count the abundance of A. besseyi in the seeds.

Development of Aphelenchoides besseyi Infestation Rate
Seeds that were indicated infested with A. besseyi were planted on sterile soil and manure at a ratio of 1:1. Planting was done in a specific seedling pot with one seed hole. The soil was watered one to two times a day depending on the weather. Observations were carried out every day until the plant reached 21 days after planting (DAP). Thirty seeds from each variety of each district were planted with 3 repetitions.
Parameters addressed in incubation time and disease incidence. Observations were made every day and the incubation time of the A. besseyi attack was recorded. Observation on nematode attack rate carried out on seeds planted in a greenhouse on the 7th, 14th and 21st DAP. The disease incidence rate was obtained by dividing the number of attacked plants with the total number of plants and then multiplied by 100%.

Data Analysis
The abundance of A. besseyi population that found in various rice seed varieties were then averaged and determined the safeness of each variety to be cultivated with the consideration that more than 30 nematodes per 100 seeds would be able to cause yield loss (Fukano, 1962), thus the seeds are not safe to plant. If A. besseyi nematode was found less than 30 nematodes per 100 seeds, it means that the seed variety is still safe to be planted. Data on the infestation rate per week (data per variety and time) were analyzed using a one-way analysis of variance (ANOVA) (α = 0.05). If there were real differences, the data will further be tested using Duncan's New Multiple Range Test (DMRT) (α = 0.05).

RESULTS AND DISCUSSION
Detection of A. besseyi in seeds are difficult to be done with bare eyes due to not all symptomatic seeds were infested by the nematode. A. besseyi which was found to have slender shape, set off the mouth, short stylet, the median oval-shaped bulb that range ¾ body width ( Figure 1A), has a mucro at the end of the posterior ( Figure 1B), and overlapping esophagus.
Tables 1 and 2 explained that from 17 varieties with symptoms, only 16 varieties were infested with A. besseyi. This implies that certified seeds are not assured free from A. besseyi infestation. Similar research conducted by Diana et al. (2018) also stated that seeds from inbreeding upland rice, paddy rice, local varieties and excellent varieties of sticky rice can be infected by A. besseyi. In addition, A. besseyi also be found not only in rice seeds that show symptoms which is shape changes and bear black spots on the seed coat but also in seeds with no symptoms (Ahmad, 2017).
From 100 symptomatic seeds, there were only 1-9 seeds infested with A. besseyi. Nematode populations found in 100 symptomatic seeds were ranging from 0 in the R3 Situbagendit to 116 in Ketan varieties. Fukano (1962) determined 30 nematodes per 100 seeds as the limit population of A. besseyi to have cause yield loss. Because A. besseyi infestation in the rice seeds observed in this research were less than 30 nematodes per 100 seeds, this revealed that seeds were still safe to be cultivated, except for R1 WAB, R1 Situbagendit, Ketan, and IR 64 ( Table 2). The incubation time of A. besseyi attacking various varieties was varied from the 3rd to the 8th DAP. This is not much different from EPPO (2013) which stated that the first symptoms of A. besseyi attack on rice can be observed on the 6th DAP (Table 2).
A. besseyi provide typical symptoms at the beginning of growth, that is chlorosis at the tip of young leaves with a length of 1 cm (Figure 2A), then the chlorosis expands to 2 cm in the first week ( Figure 2B), and reach 5 cm in the second week ( Figure 2C). Then the tips of the leaves dried and wrinkled ( Figure 2D) in the third week. The same results were also obtained by Sari (2017) and Azizah (2017) who mentioned the symptoms due to the infestation of A. besseyi were dried buds, curled, and wrinkled. A. besseyi attacks on rice plants causing stunted plant cell growth which lead to the absence of chloroplasts and hence leads to chlorosis of leaf buds. This nematode also damaged phloem tissue by disrupting the distribution of nutrients in plants which could be seen in interfered plant growth (Fortuner & Williams, 1975).  Other symptoms of the attack are flag leaf bending, panicles emerging inhibition, reducing rice grains, sterile flowers, seed distortion, and weight reduction in 1000 seeds. In the fields, economic losses of 0-70% depend on variety, year, and country (Todd & Atkins, 1958cit. Teng et al., 1994Yoshii & Yamamoto, 1995;Todd & Atkins, 1958cit. Teng et al., 1994. The development of the symptoms started from the leaf buds to the base. White tip disease will expand followed by necrosis. When the seeds were sown,    A. besseyi anabiosis immediately and attracted by the meristematic. At the initial growth, A. besseyi will be in the leaf fronds in a small population. This nematode will parasite the host plants ectoparasitically around the apical meristem area (Luc et al., 1990). The development of the infestation rate of A. besseyi on plant varieties increased every week. The highest attack in the first week occurred on the R2 Situbagendit variety (20.93%) although they were not different from the Inpari 33. The incidence strongly increased in the second week with the highest infestation that occurred in the R2 Situbagendit where the value was nearly equal to the 3rd-week attack in several varieties. The peak of the attack occurred in the third week, although the number of attacks in each variety was the same except in the R1 Situbagendit, R3 Situbagendit and R2 IR 64 varieties, where the number of attacks in the second week was higher than the number of attacks in the third week (Table 3).
The increasing percentage of A. besseyi infestation rate in various rice seed varieties due to the transmission of nematodes from one plant to another. The same result also obtained by Efendi (2016) and Mahdavian & Javadi (2012) which stated that transmission between plants can occur when planted in the same area. Infested symptoms decreased in Situbagendit R1, R2 IR 64, and Situbagendit R3 presumably because the plant was able to regenerate immediately hence the plant grows normal again. Masked symptoms (plants not showing any symptom even infected by A. besseyi) can also occur in infected plants. The number of nematodes did not affect the symptoms that appeared in plants. A high number of the nematode population does not always cause symptoms in plants but can lead to yield loss (Feng et al., 2014).