Sand Particle Size and Phosphorus Amount Affect Rhizophagus irregularis Spore Production Using In Vitro Propagated Spore as a Starter Inoculum in Rhizosphere of Maize (Zea mays) Plantlets

Microbial inoculants, particularly arbuscular mycorrhizal (AM) fungi, have great potential for sustainable crop management. In this study, monoxenic culture of indigenous R. irregularis was developed and used as a tool to determine the minimum phosphorus (P) level for maximum spore production under the in vitro conditions. This type of starter AM fungal inoculum was then applied to an in vivo substrate-based mass-cultivation system. Spore production, colonization rate, and plant growth were examined in maize (Zea mays L.) plant inoculated with the monoxenic culture of R. irregularis in sand graded by particle size with varying P levels in nutrient treatments. In the in vitro culture, the growth medium supplemented with 20 µM P generated the maximum number of spores (400 spores/mL media) of R. irregularis. In the in vivo system, the highest sporulation (≈500 spores g−1 sand) occurred when we added a half-strength Hoagland solution (20 µM P) in the sand with particle size between 500 µm and 710 µm and omitted P after seven weeks. However, the highest colonization occurred when we added a half-strength Hoagland solution in the sand with particle sizes between 710 µm and 1000 µm and omitted P after seven weeks. This study suggests that substrate particle size and P reduction and regulation might have a strong influence on the maximization of sporulation and colonization of R. irregularis in sand substrate-based culture.

Phosphorus is a critical factor of the in vitro monoxenic culture method for a wide range of arbuscular mycorrhizal fungi culture collections

Establishing an effective way to propagate a wide range of arbuscular mycorrhizal (AM) fungi species is desirable for mycorrhizal research and agricultural applications. Although the success of mycorrhizal formation is required for spore production of AM fungi, the critical factors for its construction in the in vitro monoxenic culture protocol remain to be identified. In this study, we evaluated the growth of hairy roots from carrot, flax, and chicory, and investigated the effects of the phosphorus (P) concentration in the mother plate, as well as the levels of P, sucrose, and macronutrients in a cocultivation plate with a hairy root, amount of medium of the cocultivation plate, and location of spore inoculation, by utilizing the Bayesian information criterion model selection with greater than 800 units of data. We found that the flax hairy root was suitable for in vitro monoxenic culture, and that the concentration of P in the cocultivation plate was a critical factor for mycorrhizal formation. We showed that an extremely low concentration of P (3 μM) significantly improved mycorrhizal formation for AM fungi belonging to the Glomerales order, while a high concentration of P (30 μM) was suitable for Diversisporales fungi. Therefore, we anticipate that the refining the P concentration will contribute to future culture collections of a wide range of AM fungi.

Establishment of new monoxenic culture systems for root-knot nematodes, Meloidogyne spp., on axenic water spinach roots

To establish optimal monoxenic culture systems for Meloidogyne spp., in vitro axenic culture methods of water spinach and tomato roots and monoxenic reproduction of the nematodes were assessed on six different media (MS-I to MS-VI), based on the standard Murashige and Skoog (MS) medium. The concentrations of the major elements of MS medium were amended, and the main differences among these media were concentrations of iron salts, hormone and ammonium nitrate in the macroelements. Both water spinach and tomato successfully produced roots on MS-I to MS-V media (with ammonium nitrate), but not on MS-VI (without ammonium nitrate). Water spinach produced many more roots than tomato, especially on MS-I (without hormones). Meloidogyne incognita was successfully established on in vitro roots of the two plants on MS-I to MS-V media. It was also possible to culture M. javanica and M. arenaria on water spinach root on MS-I medium. In addition, water spinach root tissues could be maintained for 8 months on MS-II and MS-V media.

Establishing monoxenic culture of arbuscular mycorrhizal fungus Glomus intraradices through root organ culture

Arbuscular mycorrhizal fungi are soil fungi distributed worldwide, forming symbiosis with most of the vascular plants for their growth and survival, which is used for sustainable agriculture and ecosystem management. This study investigated the establishment of monoxenic cultures of Glomus intraradices in association with transformed carrot hairy root. The G.intraradices spores were isolated from sugarcane rhizosphere by wet sieving and decanting technique and propagated in open pot culture. Transformation in to carrot hairy root was done using Agrobacterium rhizogenes. Surface sterilization of G.intraradices spores co-cultured with transformed carrot hairy root in Modified Strulla and Romand (MSR) medium was found the host root growth as well as for germination AM spores. After three months of incubation in dark condition, significant production of extensive hyphal growth on MSR medium and an average of 8500-9000 spores per petri dish was observed. The in vitro inoculum exhibited higher potential of root colonization due to numerous intraradices mycelium with extensive spore load. The produced monoxenic inoculum can be used in place of traditional system where it has a advantage of producing contaminant free propagulas. Thus the monoxenic culture system, a powerful tool, of AM sporulation, can be used for the mass production of monoxenic inoculum of AM fungi besides studying its biology.

POTENTIALLY PATHOGENIC FREE-LIVING AMOEBAE IN SOME FLOOD-AFFECTED AREAS DURING 2011 CHIANG MAI FLOOD

SUMMARY The survey was carried out to investigate the presence of potentially pathogenic free-living amoebae (FLA) during flood in Chiang Mai, Thailand in 2011. From different crisis flood areas, seven water samples were collected and tested for the presence of amoebae using culture and molecular methods. By monoxenic culture, FLA were detected from all samples at 37 °C incubation. The FLA growing at 37 °C were morphologically identified as Acanthamoeba spp., Naegleria spp. and some unidentified amoebae. Only three samples (42.8%), defined as thermotolerant FLA, continued to grow at 42 °C. By molecular methods, two non-thermotolerant FlA were shown to have 99% identity to Acanthamoeba sp. and 98% identity to Hartmannella vermiformis while the two thermotolerant FLA were identified as Echinamoeba exundans (100% identity) and Hartmannella sp. (99% identity). This first report of the occurrence of FLA in water during the flood disaster will provide information to the public to be aware of potentially pathogenic FLA.

Susceptibility of Acanthamoeba to multipurpose lens-cleaning solutions

The present study investigated the susceptibility of Acanthamoeba spp. trophozoites to two multipurpose systems for cleaning and maintenance of contact lenses. Three strains of trophozoites from the ATCC (A. castellani T4, A. castellani Neff, and A. polyphaga) and two Acanthamoeba isolates obtained from swimming pools (PT5 and PO1) were placed in monoxenic culture. To test their survival in cleaning solutions for contact lenses, the trophozoites were exposed for 4 and 24 h to two multipurpose solutions (A and B), and were then inoculated into a new monoxenic culture. Amoebic growth on the plates was observed after 72 h of incubation. Trophozoites from all three ATCC strains and one isolate from a swimming pool (PO1) grew in all plates after 4 h of exposure to solutions A and B. After 24 h, the ATCC strains and the PO1 isolate showed growth in most of the plates treated. Only the PT5 isolate showed susceptibility to both solutions over the time intervals tested. The two solutions were not completely effective against most strains and isolates over the time intervals tested. These results are important, since species of Acanthamoeba are widely distributed in the environment and are potential agents of eye pathologies.