Endophytic Colletotrichum species from Dendrobium spp. in China and Northern Thailand

Species of Colletotrichum are commonly found in many plant hosts as pathogens, endophytes and occasionally saprobes. Twenty-two Colletotrichum strains were isolated from three Dendrobium species – D. cariniferum, D. catenatum and D. harveyanum, as well as three unidentified species. The taxa were identified using morphological characterisation and phylogenetic analyses of ITS, GAPDH, ACT and ß–tubulin sequence data. This is the first time to identify endophytic fungi from Dendrobium orchids using the above method. The known species, Colletotrichum boninense, C. camelliae-japonicae, C. fructicola, C. jiangxiense and C. orchidophilum were identified as fungal endophytes of Dendrobium spp., along with the new species, C. cariniferi, C. chiangraiense, C. doitungense, C. parallelophorum and C. watphraense, which are introduced in this paper. One strain is recorded as an unidentified species. Corn meal agar is recommended as a good sporulation medium for Colletotrichum species. This is the first report of fungal endophytes associated with Dendrobium cariniferum and D. harveyanum. Colletotrichum camelliae-japonicae, C. jiangxiense, and C. orchidophilum are new host records for Thailand

Ultrasensitive determination of absolute mRNA amounts at attomole levels of nearly identical plant genes with high-throughput mass spectrometry (MassARRAY)

Detection of very small amounts of RNA based on microdissection of plant tissue is essential for modern plant biology. Mass spectroscopy technology (MassARRAY) based on Sequenom(TM) instrumentation was adapted to determine quickly and in a high-throughput fashion (by multiplexing) the absolute amounts of mRNA of closely related soybean genes. A sensitivity of 0.1 amol (10(-19)) was achieved, representing as few as 1,000 mRNA molecules. This methodology eliminates the use of housekeeping genes as reference standards and has multiple applications for plant functional genomics, such as the monitoring of individual expression of paralogous genes at ultra-low expression levels and/or in extremely small tissue samples

Signal sensing for induction and autoregulation of nodulation in soybean

Nodulation of legumes is induced by lipo-oligosaccharide Nod factors (NF) produced by rhizobia. We have cloned the soybean NF receptors GmNFR1 and GmNFR5) and verified their assignment by complementation of mutant alleles. Overexpression leads to increased nodulation and apparent diminishment of autoregulation of nodulation (AON). AON is controlled in part by a leucine-rich repeat receptor kinase gene (GmNARK) with structural similarities to the CLAVATA1 of Arabidopsis. Mutation results in abundant nodulation caused by the loss of a yet-undefined negative nodulation repression system involving root-leaf-root signalling. Autoregulation check-points differ between legumes; it appears that indeterminate nodulators such as Medicago and pea block early infection events. In contrast soybean, a determinate nodulator, predominantly regulates nodule ontogeny through blockage of primordium advancement. Expression of GmNARK was restricted to vascular cells of leaves, roots and stems. Sensing for autoregulation initiation was investigated by approach grafts using non-nodulation mutants as sensor, and wild type as reporter. Sensing required GmNFR5, part of the NF receptor complex. Root hair curling and infection threat formation were not essential suggesting the existence of an activated state as a consequence of NF perception, which leads to induction of root hair colonization, cortical cell division and AON

Nodulation control in legumes

Nodulation and concomitant symbiotic nitrogen fixation are critical for the productivity of the legume, yielding food, feed and fuel. The nodule number in legumes is regulated by numerous factors including the number and efficiency of the interacting Rhizobium bacteria and abiotic stresses as well as endogenous processes involving phytohormones, nodulation reception systems and autoregulation of nodulation (AON; Kinkema et al., 2006). The original discovery of the AON-controlling LRR receptor kinases, GmNARK/ LjHAR1/MtSUNN, which is active in leaf tissue of several legu-mes, now has led to an analysis of the mechanism underlying the signal transduction

Functional genomic analysis of systemic cell division regulation in legumes

Legumes develop root nodules from pluripotent stem cells in the rootpericycle in response to mitogenic activation by a decorated chitin-likenodulation factor synthesized in Rhizobium bacteria. The soybean genes encoding the receptor for such signals were cloned using map-based cloning approaches. Pluripotent cells in the root pericycle and the outer or inner cortex undergo repeated cell divisions to initiate a composite nodule primordium that develops to a functional nitrogen-fixing nodule. The process itself is autoregulated, leading to the characteristic nodulation of the upper root system. Autoregulation of nodulation (AON) in all legumes is controlled in part by a leucine-rich repeat receptor kinase gene (GmNARK). Mutations of GmNARK, and its other legume orthologues, result in abundant nodulation caused by the loss of a yet-undefined negative nodulation repressor system. AON receptor kinases are involved in perception of a long distance, root-derived signal, to negatively control nodule proliferation. GmNARK and LjHAR1 are expressed in phloem parenchyma. GmNARK kinase domain interacts with Kinase Associated Protein Phosphatase (KAPP). NARK gene expression did not mirror biological NARK activity in nodulation control, as q-RT-PCR in soybean revealed high NARK expression in roots, root tips, leaves, petioles, stems and hypocotyls, while shoot and root apical meristems were devoid of NARK RNA. High through-put transcript analysis in soybean leaf and root indicated that major genes involved in JA synthesis or response are preferentially down-regulated in leaf but not root of wild type, but not NARK mutants, suggesting that AON signaling may in part be controlled by events relating to hormone metabolism. Ethylene and abscisic acid insensitive mutants of L. japonicus are described. Nodulation in legumes has significance to global economies and ecologies, as the nitrogen input into the biosphere allows food, feed and biofuel production without the inherent costs associated with nitrogen fertilization [1]. Nodulation involves the production of a new organ capable of nitrogen fixation [2] and as such is an excellent system to study plant – microbe interaction, plant development, long distance signaling and functional genomics of stem cell proliferation [3, 4]. Concerted international effort over the last 20 years, using a combination of induced mutagenesis followed by gene discovery (forward genetics), and molecular/biochemical approaches revealed a complex developmental pathway that ‘loans’ genetic programs from various sources and orchestrates these into a novel contribution. We report our laboratory’s contribution to the present analysis in the field