Earthworm abundances in endophyte-infected tall fescue pastures in Northwest Arkansas

The ecology of organisms that co-evolve within an ecosystem is likely to be distinct from that involving organisms recently introduced into an area. To better understand the relationship of earthworms with endophyte-infected tall fescue, earthworms in novel and toxic endophyte-infected tall fescue pastures were enumerated and identified as adults or juveniles. We hypothesized that differences in endophyte infection of the fescue would influence earthworm abundances. Earthworms in two toxic and two novel endophyte-infected tall fescue fields in Fayetteville, Ark., were sampled weekly from January through July 2007. Each type of endophyte-infected pasture was established in 1997 and 2003. Sampling was carried out utilizing a physical dig-and-sort extraction method. Although variable, sampling time was a significant factor in the number of adult and juvenile worms collected. Adult earthworm abundances showed a seasonal trend of declining numbers from winter to summer, while juvenile worms showed an increase from winter to summer. Previous studies have shown that endophyte infection of plants can impact soil organisms. In this study, type of fungal endophyte infection did not appear to impact earthworm abundances; therefore, use of novel endophyte-infected fescue in a pasture is not expected to have an impact on the ecology of earthworms

Dual mutualistic associations in sainfoin (Onobrychis viciifolia Scop.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Agronomy at Massey University

Recent studies established that many legumes, when infected with the appropriate Rhizobium spp. and arbuscular fungi, nodulated better and exhibited greater dinitrogen fixation than plants infected with only the rhizobia. A similar study, therefore, was carried out in a glasshouse using sainfoin (Onobrychis viciifolia Scop.), a legume that is rapidly gaining recognition as a potential forage crop in New Zealand and other parts of the world. Pre-germinated seeds (cv. Fakir) were planted in sterilized soils and incubated with an effective Rhizobium spp. (strain NZP 5301), a mixture of endophytes (Gigaspora magarita Becker & Hall, Glomus fasciculata (Thax. sensu Gerd.) Gerdemann & Trappe and Glomus tenuis (Greenall) Hall), or both eht rhizobia and endophytes. The experiment also included a control, without any inoculation. Endophyte infection, nodulation and dinitrogen fixation, total nitrogen and phosphorus concentrations, and plant growth and development were determined on eleven sequential samplings over about twenty weeks, up to the stage of green inflorescence. Arbuscular mycorrhiza formation did not occur with the first endophyte inoculation, containing Gigaspora magarita Becker & Hall, even after 93 days of growth. This is probably because the inoculum used consisted of a low quantity of viable spores and mycelia. The second inoculation, containing the three endophyte species, produced only a low degree of infection between day 115 and 137, possibly because the extensive root lignification and relatively higher root phosphorus concentration (0.50%) restricted fungal invasion and establishment within the root cortex. Mycorrhiza formation did not increase phosphate uptake, improve nodulation and dinitrogen fixation, or increase plant growth. This is due probably to the already well-developed root systems that were efficiently exploiting the small soil volume within the bags. Rhizobia-inoculated plants produced more nodules, larger nodules and consequently, a greater nodule dry weight than the uninoculated plants. The nodules produced in the inoculated plants were red instead of green as in the uninoculated plants, and exhibited a greater dinitrogen fixation. As a result, these inoculated plants contained a higher concentration of shoot, root and nodule nitrogen, and a greater dry weight accumulation in the shoots and nodules. The shoot and nodule phosphorus concentrations, however, were lower in the rhizobia-inoculated than in the uninoculated plants due to the greater amount of shoot and nodule tissues which caused a dilution effect. These rhizobia effects on nodulation and dinitrogen fixation, nitrogen and phosphorus concentrations, and plant growth and development became more prominent with time. The relatively higher nodule phosphorus concentration when compared with the shoot and root phosphorus concentrations suggests that phosphorus was presumably required in large quantities by the dinitrogen-fixing system

Infection by a foliar endophyte elicits novel arabidopside-based plant defence reactions in its host, Cirsium arvense

Endophytic fungi live asymptomatically within plants. They are usually regarded as non-pathogenic or even mutualistic, but whether plants respond antagonistically to their presence remains unclear, particularly in the little-studied associations between endophytes and nong-raminoid herbaceous plants. We investigated the effects of the endophyte Chaetomium cochlioides on leaf chemistry in Cirsium arvense. Plants were sprayed with spores; leaf material from both subsequent new growth and the sprayed leaves was analysed 2 wk later. Infection frequency was 91% and63% for sprayed and new growth, respectively, indicating that C. cochlioides rapidly infects new foliage. Metabolomic analyses revealed marked changes in leaf chemistry with infection, especially in new growth. Changes in several novel oxylipin metabolites were detected, including arabi-dopsides reported here for the first time in a plant species other than Arabidopsis thaliana,and a jasmonate-containing galactolipid. The production of these metabolites in response to endophyte presence, particularly in newly infected foliage, suggests that endophytes elicit similar chemical responses in plants to those usually produced following wounding, herbivory and pathogen invasion. Whether en-dophytes benefit their hosts may depend on a complex series of chemically mediated interactions between the plant, the endophyte, other microbial colonists and natural enemies

Controlling sap-sucking insect pests with recombinant endophytes expressing plant lectin

We developed a novel pest management strategy, which uses endophytes to express anti-pest plant lectins. Fungal endophyte of Chaetomium globosum YY-11 with anti-fungi activities was isolated from rape seedlings, and bacterial endophytes of SJ-10 (Enterobacter sp.) and WB (Bacillus subtilis) were isolated from rice seedlings. Pinellia ternate agglutinin gene was cloned into SJ-10 and WB for expression by a shuttle vector, and YY-11 was mediated by Agrobacterium tumefaciens. Positive transformants were evaluated using PCR and Western blot assay. Recombinant endophytes colonized most of crops, and resistance of rice seedlings, which were inoculated with the recombinant endophytic bacteria, to white backed planthoppers was dramatically enhanced by decreasing the survival and fecundity of white backed planthoppers. Rape inoculated with recombinant endophytic fungi significantly inhibited the growth and reproduction of aphids. Recombinant endophytes expressing PTA may endow hosts with resistance against sap-sucking pests

Development of the [beta]-glucuronidase reporter gene system to study Acremonium endophyte interactions with perennial ryegrass : a thesis presented in fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University

A transformant of the fungal endophyte Acremonium lolii, strain Lp19, containing the gusA gene under the control of the constitutive Pgpd promoter was generated, and assigned the name KS1. Analytical digests and Southern hybridisation showed that this transformant contained a single chromosomally integrated copy of the gusA gene. The transformation frequency of Lp19 was found to be very low, and attempts to increase the transformation frequency were unsuccessful. KS1 was used to artificially infect seedlings of several different genotypes of Lolium perenne, all of a single cultivar, 'Nui'. These seedlings were grown into mature plants, and the endophytically produced GUS enzyme was extracted from individual plant tissues. Assays were performed on the enzyme extracts, and the levels determined were used as a measure of endophyte metabolic activity. Alterations of the gusA gene in some plants was detected by Southern hybridisation. One alteration was found to result in loss of GUS activity, the other did not appear to alter gusA expression. Levels of transformed endophyte GUS activity were initially compared between clonal plant material of a single genotype. Statistical analysis revealed that no significant differences were detectable for a particular tissue between the different plants. This showed that plant material of identical genotype could be pooled for analysis without the pooling of the individual plants having an affect on the outcome of the analysis. Next, levels of the transformed endophyte GUS activity were compared between genetically diverse perennial ryegrass plants of cultivar 'Nui'. Significant differences in GUS activity were detected in most tissues tested between the different genotypes, with only the most mature tissue displaying no detectable differences. Finally, a single plant of each of two individual genotypes was divided into several clonal plants, and the resulting mature plants were pooled in their genotypes for analysis of GUS, peramine, ergovaline and lolitrem B levels. The F test was not particularly sensitive in this experiment, and only one major difference between genotypes could be detected. Despite this, some trends emerged which were found to be consistent with those found in other studies. Metabolic activity and peramine levels were shown to be highest in the leaf sheath tissue, with levels generally decreasing with increasing tissue age. Lolitrem B was found to be highest in leaf sheath tissue also, but with levels increasing in general with tissue age. Ergovaline levels were very low in all tissues. The results presented show the potential of the use of the GUS reporter gene system to study endophyte gene expression in planta, and pooling of plants can be carried out to allow simultaneous study of toxin expression

Graphania mutans (Walker) and Acremonium lolii (Latch) : the relationship between an insect herbivore and a fungal endophyte of perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Zoology at Massey University

In examining the relationship between Graphania mutans (Walker) (Lepidoptera: Noctuidae) and perennial ryegrass infected with the fungal endophyte Acremonium lolii (Latch in press), the biology of G. mutans was investigated. Two types of larval development are identified: 'fast-track' larvae develop more rapidly through fewer instars and grow much larger than 'slow-track' larvae when reared on both artificial diet and perennial ryegrass. The complexity of Graphania speciation is discussed. The presence of endophyte is shown to confer on perennial ryegrass resistance to G. mutans larvae in the laboratory, with strong antixenosis and possible antibiosis effects exhibited. Feeding preference tests show that neonate and sixth instar fast-track larvae significantly prefer excised endophyte-free ryegrass to endophyte-infected leaves. The effects of endophyte on the development of fast-track and slow-track larvae are to decrease larval weight, head capsule width, and the number of successful pupations. The same methods were used to determine the effects of peramine (an antifeedant compound for Argentine stem weevil extracted from endophyte-infected perennial ryegrass) on fast-track G. mutans larvae. Incorporated into artificial diet at 10ppm, peramine has no affect on neonate and sixth instar larval feeding preference. peramine does affect larval development, causing reduced larval weight, delayed pupation, and, increased mortality. The role of peramine in endophyte-induced resistance, and the possible adaptive significance for perennial ryegrass of endophyte infection is considered. The interactions between G. mutans, endophyte and perennial ryegrass within the pasture ecosystem are discussed, and suggestions and hypotheses presented for future investigation

Cloning and characterisation of two subtilisin-like protease genes from Neotyphodium lolii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University

PCR amplification of Neotyphodium lolii genomic DNA with degenerate primers detected two different sequences with homology to subtilisin-like proteases. These two PCR products were used to screen a N. lolii Lp19 genomic library. The prt1 gene was isolated by screening the genomic library with the GH30 PCR product. This gene encodes a putative peptide of 434 amino acids that is most similar to subtilisin-like proteases from Aspergillus sp. The prt1 gene contained a single intron, which was in a position conserved with other fungal genes. 3'RACE was used to determine the polyadenylation site for the prt1 gene. Repetitive DNA was a feature of both the 3' untranslated region (UTR) and sequences downstream of the prtl gene. Within the 3' UTR, a complex microsatellite was found extending over 50 base pairs. Downstream of the gene, a minisatellite locus of 360 base pairs in size was found, consisting of 40 copies of a 9 base pair AT-rich repeat. Expression of prt1 was examined in cultures with various types of carbon and nitrogen sources. Although no conclusive results could be drawn, the type of carbon and nitrogen available did have some effect on prt1 expression. Repression of prt1 expression was only observed in media supplemented with sucrose and glutamate. A 500 bp fragment from the prt1 promoter was introduced into the vector pFunGus to create a translational fusion with gusA. This vector, pMM9, was transformed into Penicillium paxilli. Although transformation frequencies were low, the transformants obtained appeared to be stable for hygromycin resistance. Expression of GUS was observed in seven out of twelve of the stable transformants. This showed that the promoter fragment in pMM9 was sufficient for expression of GUS in a heterologous system. The prt2 gene was isolated by screening a genomic library with the GH3 PCR product. Partial sequence has been obtained for the prt2 gene. The prt2 gene contains at least three introns, the first of which is conserved with prt1. From the sequence obtained, prt2 encodes a peptide with strong similarity to subtilisin-like proteases from Metarhizium anisopliae, a fungal pathogen of insects