Application of Arbuscular Mycorrhizal Fungi for Growth Enhancement of Tectona grandis Linn.f. and Aquilaria crassna Pierre ex Lec.

Abstract

Arbuscular mycorrhizal (AM) fungal diversity was assessed in 8 plantations of Aquilaria crassna and Tectona grandis, endangered tree species and quality timber, respectively. Microscopic analyses were used to assess root colonization percentage and number of AM fungal morphotypes in soil samples. Soil samples were analyzed for chemical properties and glomalin–related soil protein content. Tectona grandis roots had greater AM fungal colonization than A. crassna roots, 77–91% vs. 44–79% of root length, respectively. A total of 29 AM fungal morphotypes were found, representing four families Glomeraceae (46.82%), Acaulosporaceae (36.38%), Claroideoglomeraceae (10.70%), and Gigasporaceae (6.10%). The lowest colonization percentage in A. crassna was correlated with the lower soil pH in studied areas. AM fungus colonization of T. grandis roots was positively correlated with spore density and negatively correlated with easily extractable Bradford reactive soil proteins and soil organic carbon. A positive correlation was observed between AM fungal spore density and total and easily extractable Bradford reactive soil proteins in rhizosphere soils of A. crassna, while in T. grandis spore density decreased when soil organic carbon and easily extractable Bradford reactive soil proteins increased.

The study of arbuscular mycorrhizal (AM) fungus community structure in roots and rhizosphere soils of A. crassna and T. grandis from plantations in Thailand was investigated to understand whether AM fungal communities present in roots and rhizosphere soils vary with host plant species and study sites. Terminal restriction fragment length polymorphism complemented with clone libraries revealed that AM fungal community composition in A. crassna and T. grandis were similar. A total of 38 distinct terminal restriction fragments (TRFs) were found, 31 of which were shared between A. crassna and T. grandis. AM fungal communities in T. grandis samples from different sites were similar, as were those in A. crassna. The estimated average minimum numbers of AM fungal taxa per sample in roots and soils of T. grandis were at least 1.89 vs. 2.55, respectively, and those of A. crassna were at least 2.85 vs. 2.33 respectively. The TRFs were attributed to Claroideoglomeraceae, Diversisporaceae, Gigasporaceae and Glomeraceae. The Glomeraceae were found to be common in all study sites. Specific AM taxa in roots and soils of T. grandis and A. crassna were not affected by host plant species and sample source (root vs. soil) but affected by collecting site. In preliminary experiment of AM fungal inoculation on growth enhancement of A. crassna and T. grandis, T. grandis plantlets had the best growth response to AM fungal spore inoculation than uninoculated plantlets. Tectona grandis plantlets inoculated with Claroideoglomus etunicatum PBT03 had highest height followed by C. etunicatum NNT10, E. colombiana and uninoculated plantlets, respectively. There were no significant differences in the number of leaf and width of leaf among the treatments. In A. crassna, there was no significant difference in height, number of leaf, and wideness of leaf between inoculated and uninoculated plantlets between treatments. In confirmation experiment, T. grandis plantlets had greatest height when inoculating with C. etunicatum PBT03 followed by C. etunicatum NNT10, F. mosseae RYA08, E. colombiana CMU05, and uninoculate control, respectively. The in vitro inoculation method resulted in the regrowth of AM fungal hyphae from initial inoculating root organ culture of G. intraradices and F. mosseae RYA08, and spore germination of C. etunicatum NNT10 and C. etunicatum PBT03. Tectona grandis plantlets inoculated with F. mosseae RYA08 had highest plant height follow by G. intraradices, C. etunicatum NNT10, C. etunicatum PBT03, and uninoculated plant, respectively. Inoculation with G. intraradices was affected on shoot wet weight but gave low shoot dry weight inferior to F. mosseae RYA08. There was no significant different of the number of leave and root wet weight between inoculated and uninoculated plantlets. AM fungal spores of C. etunicatum NNT10, C. etunicatum PBT03 and F. mosseae RYA08 were propagated using different culture materials (sterile sandy soil by itself or mixed 1:1 (v/v) with clay-brick granules, rice husk charcoal, or vermiculite) and host plants (Mimosa invisa, Sorghum bicolor or Zea mays). Results indicated that root colonization and number of spores of each AM fungus isolate were affected by host plant and substrate. Total AM fungal spores and percentage of root length colonized were highest when cultured with Z. mays (3,690 spores 100 cm−3 and 65% root length colonized) and when vermiculite was used as diluent (3,612 spores 100 cm−3 and 63% root length colonized). Inocula produced in the first experiment were used to evaluate the efficiency of locally-available leaf litter compost as a component of media for on–farm inoculum production. Subsequent on-farm production of mycorrhizal fungus was propagules with Allium cepa, Paspalum notatum, Tagetes patula and Z. mays. The results showed that spore production with Z. mays in leaf litter compost mixed with vermiculite was considerably higher than that with other host plants. For in vitro production, pre-germinated spore experiment showed germination of C. etunicatum NNT10, C. etunicatum PBT03, F. mosseae RYA08, and G. intraradices both on M and MSR medium. Germination percentages of most spore species tend to be higher in MSR medium measuring from hyphal length of individual germinated spore and percentage of spore germination. Germination percentage of G. intraradices was higher than those in other species. Glomus intraradices and F. mosseae RYA08 hyphae were spreaded throughout the Petri plates and produced new spores successfully after 14 and 24 days of inoculation, respectively. Claroideoglomus etunicatum PBT03 and C. etunicatum NNT10 were produced only hyphae but not spread throughout the media and no new spores were produced at 5 months after inoculation. The denaturing gradient gel electrophoresis experiment (DGGE) was performed to investigate selected AM fungal spores colonization in inoculated T. grandis plantlets.

One year–old after AM fungal inoculation of T. grandis roots were used to extract DNA and nested amplified with AM fungal specific primers, AML1–AML2 and NS31–GC/AM1. Analysis of AM fungal colonized root by polymerase chain reaction–DGGE was performed in duplicate to check specificity of primers used in this experiment. No distinguishable difference in DGGE pattern was observed between two replicates of polymerase chain reaction (PCR) production after nested PCR. The DGGE analysis of the NS31–GC/AM1 primer products yielded banding patterns within the range of 10–18% denaturant. Amplified DNA from reference fungal spores yielded distinguishable dominant band with sharp and intense DGGE bands within the range of 10–12% denaturant. Dominant DGGE of one year-old AM fungal inoculated T. grandis roots were also showed in those denaturing gradient ranging from 2–3 bands even in uninoculated roots. The DGGE bands from reference spores and T. grandis roots which immigrated into the same denaturing gradient were reamplified and digested with restriction enzymes, HinfI and Hsp92II to confirm certain AM fungal species inside inoculated roots. The result showed that restriction fragment length polymorphism (RFLP) pattern could not identify the different of certain AM fungal species that have been inoculated into T. grandis roots. However, those patterns could estimate that the possible AM fungal species inside all roots from each treatment tend to be C. etunicatum when comparing RFLP patterns from both restriction enzyme digestions.