Biological control of clover cyst nematode Heterodera trifolii : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science (Plant Pathology) at Massey University, Palmerston North, New Zealand

Heterodera trifolii is one of the most damaging pests in New Zealand pastures and the aim of this work was to study the potential use of 'biological control' as an alternative strategy to the use of chemicals. Natural enemies, especially fungi, isolated from H. trifolii were the main organisms investigated for control of H. trifolii in this study. As a first step, more information was required on population dynamics of this nematode species in pasture soil and the possible causes of its population peaks and troughs. Soil cores were taken at fortnightly intervals from a permanent white clover/ryegrass pasture at AgResearch, Palmerston North, New Zealand, from March 1999 to March 2001 and numbers of second stage juveniles (J2), adult females and cyst stages of H. trifolii were recorded together with those of other soil nematodes. Emergence of J2 peaked in winter in both 1999 and 2000, and appeared to follow root growth, which in turn reflected soil moisture levels. Young cysts and mature females were assessed for parasitism by fungi and such fungi were isolated into pure culture as a first stage in assessing their biological control potential. Fungal genera such as Verticillium, Fusarium, Gliocladium, Paecilomyces, and Trichoderma were assessed for their pathogenicity to white clover seedlings Trifolium repens on 1.0% water agar in vitro then in sand in pots. As these fungal isolates were not pathogenic to the T. repens plants in pots, some, such as species of Fusarium, Gliocladium, and Verticillium were tested for their potential parasitism on H. trifolii in pots of sand with a view to assessing their use as biological control agents. Oatmeal was one substrate on which fungi were grown but it caused poor growth of T. repens. On the basis of these experiments, only isolate Vc6 (a Verticillium chlamydosporium isolate) consistently reduced the numbers of H. trifolii and was selected for further experiment. Vc6 was grown on a range of media such as alginate beads, bran culture alginate beads, potato dextrose broth culture alginate beads, dry soil inoculum and wheat flour/sand inoculum and it was assessed for its potential pathogenicity to H. trifolii females and cysts. Vc6 grown on alginate beads containing wheat bran significantly reduced the numbers of females and of cysts and it increased plant growth of T. repens. There were more than 100 V. chlamydosporium isolates from the two-year study so there was a need to screen the isolates for biocontrol activity as they varied in the production of chlamydospores from which eggs of adult females are parasitised. For screening experiments, clover cyst nematode was successfully cultured on 0.5% Hoagland & Knop's agar monoxenically using J2 surface-sterilised with 0.5% Hibitane in a watch glass. A number of V. chlamydosporium isolates obtained from young cysts and females of H. trifolii in the two-year field study were screened for pathogenicity to H. trifolii in monoxenic culture using T. repens seedlings on 0.5% Hoagland & Knop's agar, and in sand in pipette tips in vitro. Females of H. trifolii developed in some of the V. chlamydosporium isolate treatments and it was concluded that there were some variations in their pathogenicity to H. trifolii in the in vitro tests. In addition to screening the isolates for their parasitism to H. trifolii in vitro, variation among the isolates was investigated at the molecular level using the RAPD PCR-based technique. Cluster analysis of 10 V. chlamydosporium isolates using RAPD PCR data showed that isolate Vc6 consistently differed from other V. chlamydosporium isolates tested

Population dynamics of Meloidogyne graminicola and Hirschmanniella oryzae in a double rice-cropping sequence in the lowlands of Myanmar

The rice root-knot nematode, Meloidogyne graminicola, and the rice root nematode, Hirschmanniella oryzae, are considered potentially important nematode pathogens in lowland rice. A study was undertaken from December 2009 until December 2010 in the Ayeyarwady River Delta, the major lowland rice-producing area of Myanmar, to monitor the population dynamics of M. graminicola and H. oryzae in a naturally infested field. Root samples of the two rice varieties Yatanartoe and Taungpyan that are commonly cultivated in double rice-cropping sequences in Myanmar and represent irrigated and rainfed lowland rice varieties, respectively, were obtained for nematode analysis. During the summer-irrigated rice-growing season the root population density of second-stage juveniles (J2) of M. graminicola showed two distinct peaks – at the maximum tillering stage of the rice plants in January and at the heading stage of the rice plants in March 2010. With the onset of the monsoon rains, the J2 population densities in the roots of ratoon rice plants gradually decreased in May. During the rainfed monsoon rice-growing season, very low population densities of M. graminicola J2 were detected in the roots of rice plants, while the root population density of H. oryzae juveniles and adults showed two distinct peaks – at the maximum tillering stage of the rice plants in August and at the heading stage of the rice plants in October 2010. With the onset of the dry season, population density of H. oryzae in the roots reached the lowest density at harvest in November. Root galling caused by M. graminicola followed the same trend as the J2 population densities throughout the irrigated season. No root galls were observed during the monsoon season. Our results can be used for practical purposes aimed at a better management of both M. graminicola and H. oryzae.http://booksandjournals.brillonline.com/content/journals/10.1163/15685411-0000271

Occurrence of the rice root nematode Hirschmanniella oryzae on monsoon rice in Myanmar

During May-October 2007, soil and root samples from 539 fields were collected from 11 monsoon rice varieties in 12 regions in Myanmar. All regions surveyed and 90% of fields sampled were infested with the rice root nematode Hirschmanniella oryzae. The average H. oryzae population was 10/100 mL soil and 419/20 g roots respectively. In 6.9% of the fields sampled 50 H. oryzae/g root were found. The average root population densities were the highest (640/20 g roots) in Taungpyan variety and the lowest (155/20 g roots) in Immayebaw variety. The lowest soil and the second-lowest root populations of H. oryzae were observed in Shwethweyin which may indicate that this rice variety is less susceptible to H. oryzae. Among three cropping sequences, the highest frequency of occurrence (94%) was found in the rice-rice cropping sequence. Based on the prominence value (a combination of the frequency of occurrence and abundance) of H. oryzae, Hlaingtharyar was the most infested region where susceptible rice varieties are grown and rice-rice cropping sequence is practiced. The rice production in this region may be the most at risk of suffering important yield losses due to H. oryzae

Effect of different water regimes on nematode reproduction, root galling, plant growth and yield of lowland and upland Asian rice varieties grown in two soil types infested by the rice root–knot nematode Meloidogyne graminicola

In a screenhouse experiment, plants of the lowland rice variety Thihtayin and the upland rice variety Kone Myint 2 were grown in two soil types (clay loam and sandy loam), inoculated with 3,000 Meloidogyne graminicola second-stage juveniles (J2) per plant and from 6 weeks onwards maintained until harvest under three water regimes: permanently flooded, intermittently flooded and upland (monsoon rainfed) conditions. Both varieties were susceptible to M. graminicola infection under all three water regimes and in both soil types but differences in susceptibility were observed between the two varieties and among the treatments. The effect of water regime on the number of eggs and J2 of M. graminicola inside the roots was lower than expected: with one exception no significant effects were observed of any of the water regimes on the root population density in both rice varieties in both soil types. This observation may be explained by the delayed flooding, which started 6 days after nematode inoculation for the permanent and intermittent flooding water regimes. In both varieties and in both soil types, the root galling index was significantly lower on permanently flooded plants (< 4.5) compared with plants that had been either intermittent flooded or grown under upland conditions (≥ 5.0). The highest root galling indices were always observed on plants grown under upland conditions (7.0-8.5). Permanent flooding prevented the suppression of most plant growth and yield-contributing traits measured. Moreover, permanent flooding also prevented significant yield loss in plants of both varieties grown in the clay loam soil and in plants of variety Thihtatyin grown in the sandy loam soil. The results of our study confirm again the enormous impact M graminicola infection can have on the yield of both lowland and upland rice varieties. With the exception of one treatment, yield loss was always higher than 20% and even almost 100% (yield failure) in plants of both varieties grown in the sandy loam soil under upland conditions. Although yield losses caused by nematodes carried out under screenhouse experiments tend to result in an overestimation of these losses, the results of our screenhouse experiments show that yield losses caused on Asian rice by M. graminicola must be very high also under field conditions in the farmer's field

Comparison of the damage potential and yield loss of the rice root–knot nematode, Meloidogyne graminicola, on lowland and upland rice varieties from Myanmar

In Asia, the rice root-knot nematode, Meloidogyne graminicola, is an important pathogen of Asian rice (Oryza sativa) in most rice producing countries including Myanmar. In the first part of our study, the damage potential of M. graminicola on commonly cultivated newly released high-yielding lowland rice varieties and upland rice varieties (traditional, improved and aerobic), which are being grown in different regions in the summer-irrigated lowland and rainfed upland rice ecosystems in Myanmar, was investigated under screenhouse conditions. In the second part of our study, a field experiment was carried out in the Ayeyarwady River Delta, the major lowland rice producing area of Myanmar, to investigate the impact of M. graminicola on plant growth and yield of the same lowland rice varieties included in the screenhouse experiment in a naturally-infested farmer’s field. None of the 15 lowland varieties and none of the nine upland varieties included in the screenhouse experiments was resistant to M. graminicola. Although differences in susceptibility were observed among the lowland and upland varieties, and between the two types (lowland and upland) of rice varieties, with an Mf–eggs (multiplication factor of the second-stage juveniles without counting the eggs) ranging from 13.2 to 52.8 for the lowland varieties vs 39.8 to 108.4 for the upland varieties, all varieties included in our study can be considered as highly susceptible to M. graminicola when the nematode population densities are assessed at harvest. Also in the field experiment, all the 15 lowland varieties were susceptible to M. graminicola infection. This part of our study shows that upland rice varieties are more susceptible to M graminicola infection than lowland rice varieties. The percentage reduction in lowland and upland varieties was very similar in six out of the ten plant growth and yield-contributing traits measured. The highest differences in percentage reduction were observed for fresh root weight (41.0 vs 26.1% for the lowland and upland varieties, respectively), number of tillers per plant (29.1 vs 14.1%, respectively), percentage filled grains per plant (11.7 vs 0.8%, respectively) and filled grain weight per plant (34.7 vs 47.6 g, respectively). Within the two types of rice varieties significant differences in percentage reduction of plant growth traits between uninoculated and inoculated plants were observed among the rice varieties, so we suggest that the effect of M. graminicola infection on the different vegetative and reproductive plant growth stages of rice varieties is highly genotype-dependent and that no general conclusions can be made. In the screenhouse experiments, infection with M. graminicola caused on average a yield reduction with 31.1% in the lowland rice varieties vs 44.9% in the upland rice varieties, which indicates that in the upland varieties the higher nematode population densities per root unit (1 g) and per root system resulted in a higher yield loss compared with the lowland varieties. In the field experiment, carbofuran treatment resulted, on average, in a 16.5% increase in yield of the lowland rice varietie

New Record on the Occurrence of Cyst Nematode, Heterodera cajani Koshy, 1967 on Sesame, Sesamum indicum in Myanmar

Sesame, Sesamum indicum L. occupies for nearly half of the area sown to oilseed crops in Myanmar. It is cultivated for domestic consumption and export. During 2003, sesame cultivars, Sinyadnar3, Sinyadanar5, Magway7/9 and Khwaylayni grown in Oilseed Crop Research Farm, Department of Agricultural .Research, Magway were found to be heavily infected with Heterodera cyst nematodes. In 2004, cysts were also found from the soil samples collected from the previously infested plots. Infested field showed patches in which the plants were stunted, chlorotic, and caused wilting. When the infected plants were uprooted, curving of tap root, and red-girdling and dark browning of lateral and tap roots were observed. Lemon-shaped cysts were found attaching to the root surface. Young females were white or creamy colour whereas old cysts with eggs were tan colour. A few bullae were found on the posterior portion of the female. Larvae were vermiform with elongate conoid tail. Males were cylindrical or elongated with short tail without bursa. According to the morphological characters of the nematode and disease symptoms of sesame, the nematode was supposed to be identified as Heterodera cajani. There was no report o cyst nematode, Heterodera spp. in Myanmar. The present finding, the occurrence of Heterodera cajani on sesamum, is the first record of Heterodera spp. in Myanmar

Physician perspectives on the management of viral hepatitis and hepatocellular carcinoma in Myanmar

© 2017 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background: In Myanmar, over five million people are infected with hepatitis B virus (HBV) and hepatitis C virus (HCV). Hepatitis has been a recent focus with the development of a National Strategic Plan on Hepatitis and plans to subsidize HCV treatment. Methods: During a two-day national liver disease symposium covering HCV, HBV, hepatocellular (HCC), and end-stage liver disease (ESLD), physician surveys were administered using the automated response system (ARS) to assess physician knowledge, perceptions of barriers to screening and treatment, and proposed solutions. Multivariate logistic regression was used to estimate odds ratio (OR) relating demography and practice factors with higher provider knowledge and improvement. Results: One hundred two physicians attending from various specialty areas (31.0% specializing in gastroenterology/hepatology and/or infectious disease) were of mixed gender (46.8% male), were younger than or equal to 40 years old (51.1% 20 to 40 years), had less experience (61.6% with 10 years of medical practice), were from the metropolitan area of Yangon (72.1%), and saw \u3c10 liver disease patients per week (74.3%). The majority of physicians were not comfortable with treating or managing patients with liver disease. The post-test scores demonstrated an improvement in liver disease knowledge (9.0% ± 27.0) compared to the baseline pre-test scores; no variables were associated with significant improvement in hepatitis knowledge. Physicians identified the cost of diagnostic blood tests and treatment as the most significant barrier to treatment. Top solutions proposed were universal screening policies (46%), removal of financial barriers for treatment (29%), patient education (14%) and provider education (11%). Conclusions: Physician knowledge improved after this symposium, and many other needs were revealed by the physician input on barriers to care and their solutions. These survey results are important in guiding the next steps to improve liver disease management and future medical education efforts in Myanmar

Hepatitis knowledge score (% correct answers on knowledge-based questions).

<p>Hepatitis knowledge score (% correct answers on knowledge-based questions).</p