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Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth physiology.

Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp-tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 x 10(10) rhizobia per cm3 of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the world's most important cereal crops.

Colony Count, Microbial↗

Development and characterization of EST-derived simple sequence repeat (SSR) markers for pasture grass endophytes.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichloë isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichloë species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.

Ascomycota↗

Ultrastructure of the Alnus crispa var. mollis Fern. root nodule endophyte.

Nitrogen-fixing, field-obtained root nodules of the silky green alder were studied by transmission electron microscopy. The nodule endophyte exhibited a prokaryotic cytology and was present in two forms: the hypha(0.3-1.0mum), which was branched and septate, and the vesicle (3-5mum), which was also septate and developed at the parental hypha tip. Bacteria-like cells, previously observed in light microscopy studies, were not seen in the present work. The actinomycete-like endophyte penetrated through the host cell wall and becane enveloped by a capsular material (0.1mum), the whole being enclosed by host membranes. In some host cells, the endophyte appeared to lyse and become a mass of shrunken debris. The fine structure of the Alnus crispa var. mollis root nodule endophyte was found to be similar to that of other nonleguminous root nodule endophytes.

Bacteria↗

Ryegrass Host Genetic Control of Concentrations of Endophyte-Derived Alkaloids.

Endophytic fungi in pasture grasses produce alkaloids which affect invertebrate and vertebrate herbivores. While the competence to produce an alkaloid is a property of the fungus, the host plant may moderate fungal activity. Host genetic influence on endophyte activity was studied in perennial ryegrass (Lolium perenne L.) infected with a common strain of Neotyphodium lolii (Latch, Christensen & Samuels) Glenn, Bacon & Hanlin. Progeny seedling families of a partial diallel cross and their 12 parent clones were compared in a glasshouse experiment. Peramine and ergovaline concentrations were determined by high pressure liquid chromatography (HPLC), and intensity of endophyte infection was determined by enzyme-linked immunosorbent assay (ELISA). Concentrations of peramine and ergovaline and the amount of endophyte mycelium in plants varied between families, consistently across two glasshouse cells and (for the HPLC data) two harvests. There was no indication of any maternal effects. Host genetic control was evident in significant general combining ability effects and smaller specific combining ability effects. Parent-progeny correlation coefficients were high, and narrow-sense heritability was estimated as 0.70, 0.72, and 0.58 respectively for ergovaline, peramine, and ELISA. Further analysis indicated little interaction between loci, and no directional dominance. The three traits were correlated, indicating that 41 and 65% of the genetically controlled variation in ergovaline and peramine concentrations, respectively, was a function of mycelial mass. However, there were departures from these relationships. Host plant selection may enable development of pastures with controlled low levels of toxic but ecologically beneficial endophyte metabolites.

Journal Article↗

Thyroid function in mature horses ingesting endophyte-infected fescue seed.

OBJECTIVE: To determine whether ingestion of fescue seed infected with the endophyte Neotyphodium coenophialum would alter thyroid function in adult horses. DESIGN: Original study. ANIMALS: 4 adult mares that were not pregnant and 6 adult geldings. PROCEDURE: Thyrotropin releasing hormone stimulation tests were performed while horses received a standard diet and after infected seed (2.3 kg/d [5 lb/d]) had been fed for 1 and 2 months. Serum prolactin concentrations were measured to verify endophyte absorption. RESULTS: Serum prolactin concentrations indicated that at least 8 of 10 horses absorbed the endophyte. Baseline concentrations of thyroid stimulating hormone, total and free triiodothyronine, and total and free thyroxine and the change in hormone concentrations in response to administration of thyrotropin releasing hormone (1 mg, i.v.) were not altered by ingestion of endophyte-infected fescue seed. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that ingestion of fescue seed infected with the endophytic fungus N. coenophialum for 2 months has little effect on thyroid function in adult horses that are not pregnant.

Animal Feed↗

Effects of feeding diets containing endophyte-infected fescue seed on luteinizing hormone secretion in postpartum beef cows and in cyclic heifers and cows.

Two experiments were conducted to investigate the effect of feeding endophyte (Acremonium coenophialum)-infected fescue (Festuca arundinacea Shreb.) seed on LH secretion in postpartum beef cows and in cycling heifers and cows. In Exp. 1, spring-calving primiparous Angus cows (n = 16) were pair-fed for 75 d diets that contained endophyte-free or endophyte-infected (95%) fescue seed that contained 1.3 micrograms/g of ergovaline and 5.2 mg/g of saturated pyrrolizidines. Serial blood samples for basal and GnRH-stimulated serum LH analysis were obtained on d 7, 28, 42, and 56 of the study. The endophyte had no effect on LH secretion (basal, pulse frequency, and amplitude) or milk production. Average daily gain was decreased (P < .05) in cows that consumed infected fescue seed compared with controls (-.20 vs -.01 kg, respectively). Basal serum prolactin concentrations were reduced (P < .01) in treated compared with control cows (8.9 vs 25.4 ng/mL, respectively) on d 70. In Exp. 2, cycling Angus heifers (n = 8; age = 2 yr) and cows (n = 8; age = 4 yr) stratified by age were pair-fed for 40 d diets that contained the noninfected or the highly infected fescue seed. Estrus was synchronized by prostaglandin F2 alpha (d 18 and 28). Serial blood samples for serum LH analysis were obtained on d 28 (luteal phase) and d 30 (follicular phase). The endophyte did not affect LH (P > .28) or prolactin (P > .16) secretion, whereas ADG was decreased (P < .05) in treated compared with control animals (.32 vs .70 kg/d, respectively).

Acremonium↗

Effect of endophyte type on carcass traits, meat quality, and fatty acid composition of beef cattle grazing tall fescue.

Fourteen Hereford steers were used to compare carcass traits, meat quality, and fatty acid composition of beef from cattle grazing tall fescue infected with either wild-type (E+; n = 6) or novel, nil ergot alkaloid (AR542; n = 8) endophyte for 209 d. Average daily gain, live weight, and HCW were greater (P < 0.05) for AR542 cattle than for E+. No differences in LM color or pH were observed between AR542 and E+. Steaks from E+ cattle tended (P = 0.10) to have higher L* and b* than those from AR542 cattle at 0 d of display. Ground beef from E+ cattle also had higher (P < 0.05) L* than AR542 cattle, with no differences in a* or b* at 0 d of display. Color changes during display did not differ for both steaks and ground beef from E+ and AR542. Lipid oxidation levels increased (P < 0.05) during simulated retail display, but they did not differ between endophyte treatments. Adipose tissues from E+ cattle had a higher (P < 0.05) percentage of SFA, and a lower (P < 0.05) percentage of MUFA than adipose from AR542 cattle. Ground beef and i.m. fat had higher (P < 0.05) concentrations of SFA, MUFA, and cis-9, trans-11 isomer of conjugated linoleic acid, and lower (P < 0.05) concentrations of PUFA and PUFA:SFA ratio than s.c. fat. The n-6:n-3 fatty acid ratio did not differ among fat depots. Ergot-alkaloids were detected in s.c. adipose tissues, and alkaloid concentration was greater (P < 0.05) for E+ than AR542. Warner-Bratzler shear force values did not differ between endophyte types, but it decreased (P < 0.01) across the postmortem aging period. Conversely, sensory panel evaluation detected greater (P < 0.01) chewiness and lower (P < 0.05) juiciness for AR542 than for E+ steaks aged for 14 d. Although grazing cattle on tall fescue pastures infected with nil ergot alkaloid endophyte improved cattle performance, these results suggest that endophyte type has minor effects on carcass traits and meat quality of pasture-fed beef. Moreover, finishing cattle on tall fescue pastures showed the potential to enhance the fatty acid profile of beef from a human health perspective. Alkaloid concentration was greater (P < 0.05) in s.c. fat from E+ than AR542 (2.81 vs. 0.92 ppb; fresh-tissue basis). This is the first published report demonstrating the presence of alkaloids in beef tissues.

Adipose Tissue↗

Effects of endophyte-infected fescue on concentrations of prolactin in blood sera and the anterior pituitary and concentrations of dopamine and dopamine metabolites in brains of steers.

An experiment was conducted to determine if the decrease in circulating concentrations of prolactin in cattle consuming endophyte (Acremonium coenophialum) -infected tall fescue (Festuca arundinacea) was associated with changes in prolactin concentrations in the anterior pituitary and concentrations of dopamine (DA) and its metabolites 3,4-dihydroxyphenyl-acetic acid (DOPAC) and homovanillic acid (HVA) in the stalk median eminence (SME), preoptic area (POA) and hypothalamus (HP). Six crossbred steers that grazed high-endophyte (greater than 90% infected) fescue and four steers that grazed low-endophyte (less than 1% infected) fescue from April to September were slaughtered. Brains and pituitaries were removed and dissected. Extracts from neural tissue were analyzed for DA, DOPAC and HVA using high performance liquid chromatography/electrochemical detection. Pituitary extracts and sera from blood samples taken 5 d prior to slaughter were subjected to prolactin radioimmunoassay. Consumption of high-endophyte fescue was associated with decreased concentrations of prolactin in serum (P less than .01) and in the anterior pituitary (P = .08), decreased (P less than .05) concentrations of DA in the SME and decreased (P less than .01) concentrations of HVA in the POA and HP, but it did not influence levels of DOPAC. These results suggest that endophyte toxins may reduce prolactin synthesis and release and may alter activity of dopaminergic neurons.

3,4-Dihydroxyphenylacetic Acid↗

Effects of endophyte-infected fescue and cimetidine on respiration rates, rectal temperatures and hepatic mixed function oxidase activity as measured by hepatic antipyrine metabolism in sheep.

The effects of endophyte-infected fescue (Kentucky 31) hay and cimetidine on respiration rates, rectal temperatures and hepatic antipyrine uptakes of three mature ewes were measured in 15 experiments. Hepatic antipyrine uptake was measured as an indirect indicator of hepatic mixed-function oxidase (MFO) activity. Five experiments on each ewe were conducted during consumption of endophyte-free (one exp/ewe, control), endophyte-infected (two exp/ewe, EIF) and endophyte-infected fescue hay plus cimetidine (800 mg/d i.v., two exp/ewe, EIFC). Respiration rates increased (P less than .05) 2.6-fold after 11 d on EIF from 26 to 68 and decreased (P less than .025) to 27 breaths/min after 4 d of cimetidine treatment. Rectal temperatures increased (P less than .05) 1.1 degrees C after 8 d on EIF. Cimetidine lowered temperatures to control levels by d 4. Endophyte-infected fescue hay increased (P less than .05) hepatic antipyrine uptake 70% after 11 d, but uptake was lowered to control levels by cimetidine by d 4. Increased respiration rates and rectal temperatures in animals consuming EIF seem to be related to increased MFO activity. Cimetidine reversed some of these effects.

Alkaloids↗

Use of serum prolactin in rats as a determinant in detecting endophyte infected tall fescue seed.

The purpose of this study was to examine the effects of endophyte (Acremonium coenophialium) toxic tall fescue seed on serum prolactin, daily feed intake, and average daily weight gain in male rats. Thirty Sprague-Dawley male rats (218 g) were randomly assigned to treatments of (I) rat chow (50%) plus an endophyte-free tall fescue seed (50%), (II) same as I only restricted feed intake as III, (III) rat chow (50%) plus endophyte infected tall fescue seed (50%) and (IV) rat chow (50%) plus endophyte infected tall fescue seed (25%) plus endophyte free tall fescue seed (25%). Dry matter intake and average daily gain were lower (P less than .05) for rats fed treatment II and III than for those fed treatment I and IV, with treatment IV being lower (P less than .05) than treatment I. Body weights on days 4, 11 and 21 followed the same trend with the rats fed treatment I having higher weights at each weigh period than those on treatments II, III and IV; and those fed treatment IV having higher weights at each period than those on treatment II and III. Baseline concentrations of serum prolactin were similar among treatments. Serum prolactin concentrations were lower (P less than .05) in rats fed treatment III and IV than in those fed treatment II, and those fed treatment I were not different from all others on day 4. On day 11, rats fed treatment I, III and IV had lower (P less than .05) serum prolactin concentrations than those fed treatment II.(ABSTRACT TRUNCATED AT 250 WORDS)

Acremonium↗

Neural dopamine D2 receptors in rats fed endophyte-infected fescue seed.

To study the effect of endophyte (Acremonium coenophialum) on hypothalamic and striatal dopamine D2 receptors, male rats (n = 14/group) were pair-fed diets containing 50% Rat Chow and 50% either endophyte-infected (E+) or noninfected (E-) fescue (Festuca arundinacea Schreb.) seed for 21 days. Concentrations of ergovaline and saturated pyrrolizidines were 1.91 micrograms/g and 2.84 mg/g, respectively in E+, and undetectable in E- fescue seed. To monitor endophyte effects, rats were weighed weekly and serum derived from trunk blood (d 21) was analyzed for prolactin. Corpus striatum and hypothalamic tissue was assayed for dopamine D2 receptors using [3H]spiperone and [125I]epidepride, respectively. The endophyte depressed (P < .06) serum prolactin concentrations. Average daily gain during the study (21 d) was depressed (P < .0043) in rats fed E+ compared to controls. The endophyte increased (P < .03) striatal D2 receptor affinity (KD = 48.70 vs 54.95 pM) with no change (P > .28) in receptor density (Bmax = 25.59 vs 28.00 pmol/mg of tissue) in E+ and E- rats, respectively. Hypothalamic D2 receptor density (Bmax = 1.79 vs 1.57 pmol/mg of tissue) and affinity (KD = 17.5 vs 17.26 pM) were not (P > .66) different between E+ and E- rats, respectively. These data suggest changes in D2 receptor binding characteristics, particularly receptor affinity, may contribute to signs of fescue toxicosis.

Acremonium↗

Endophytic vs exophytic unilateral retinoblastoma: is there any real difference?

A retrospective review of 297 cases of enucleated and histologically proven unilateral retinoblastoma with a minimum of 5-year follow-up was analyzed to see if there were any clinical or prognostic differences between endophytic and exophytic type retinoblastoma. Endophytic retinoblastoma (181 cases) was found more often than exophytic (116 cases). The following features were not associated with the clinical appearance of either endophytic or exophytic tumors: sex of patient, right vs left eye, propensity for bilateral development, initial sign or symptom, presence of rubeosis, preoperative metastasis, optic nerve invasion, orbital recurrence, survival of the patient, length of follow-up, or age at diagnosis. Three features were found that correlated with type: a disproportionately higher percentage of patients with endophytic retinoblastoma had a positive family history; a disproportionately high percentage of patients with exophytic retinoblastoma developed glaucoma; and choroidal invasion occurred significantly more often in patients who had exophytic retinoblastoma than in those who had endophytic retinoblastoma.

Eye Enucleation↗

[Establishment of symbiotic system for Anoectochilus roxburghii (Wall.) Lindl. and endophytic fungi].

OBJECTIVE: Setting up a symbiotic system for Anoectochilus roxburghii and endophytic fungi, so as to study the relationship between them and try to set up new cultural methods for A. roxburghii. METHOD: A. roxburghii and its endophytic fungi were cultured together on five kinds of media in flasks. The growth of the plantlets and fungi were observed. Symbiotic conditions were selected according to symbiotic characteristics. RESULTS: A symbiotic system for A. roxburghii and endophytic fungi was set up. The optimum composition of the system induded NH4NO3 825 mg.L-1, KNO3 950 mg.L-1, MgSO4 185 mg.L-1, and inositol 100 mg.L-1, other organic components being 2/3 times those of MS medium, sugrose 15 g.L-1, and other components being the same as those of MS medium, agar 9 g.L-1, pH 5.8. The culture was effected at 24-25 degrees C under cool white fluorescent light (150 lx) for a photoperiod of 11 hours. CONCLUSION: A. roxburghii and endophytic fungi can grow well together and form endomycorrhiza in the symbiotic system. Moreover, endophytic fungi help stimulate the growth and development of A. roxburghii.

Culture Media↗

ROOT NODULE SYMBIOSIS. I. ENDOPHYTE OF MYRICA CERIFERA L.

Silver, Warren S. (University of Florida, Gainesville). Root nodule symbiosis. I. Endophyte of Myrica cerifera. J. Bacteriol. 87:416-421. 1964.-Electron microscopy of 0.1-mu thick sections of root nodules, fixed with permanganate and embedded with methacrylate, showed that infected plant cells were filled with a mycelial endophyte. The endophyte was filamentous, 1 mu in diameter, septate, and had an enlarged, club-shaped terminus. Although structurally the endophyte strongly resembles an actinomycete, it was not isolated in pure culture on a variety of appropriate media.

Actinomycetales↗

Endophytic bacteria in Coffea arabica L.

Eighty-seven culturable endophytic bacterial isolates in 19 genera were obtained from coffee plants collected in Colombia (n = 67), Hawaii (n = 17), and Mexico (n = 3). Both Gram positive and Gram negative bacteria were isolated, with a greater percentage (68%) being Gram negative. Tissues yielding bacterial endophytes included adult plant leaves, various parts of the berry (e.g., crown, pulp, peduncle and seed), and leaves, stems, and roots of seedlings. Some of the bacteria also occurred as epiphytes. The highest number of bacteria among the berry tissues sampled was isolated from the seed, and includes Bacillus , Burkholderia , Clavibacter , Curtobacterium , Escherichia , Micrococcus , Pantoea , Pseudomonas , Serratia , and Stenotrophomonas . This is the first survey of the endophytic bacteria diversity in various coffee tissues, and the first study reporting endophytic bacteria in coffee seeds. The possible role for these bacteria in the biology of the coffee plant remains unknown.

Coffea↗

Alkaloid toxins in endophyte-infected grasses.

Grasses infected with clavicipitaceous fungi have been associated with a variety of diseases including classical ergotism in humans and animals, fescue foot and summer syndrome in cattle, and rye-grass staggers in sheep. During the last decade it has been recognized that many of these fungal infections are endophytic; a fungal endophyte is a fungus that grows entirely within the host plant. Inspection of field collections and herbarium specimens has revealed that such infections are widespread in grasses. The chemistry associated with these grass-fungal interactions has proved to be interesting and complex, as each grass-fungal pair results in a unique "fingerprint" of various alkaloids, of which some are highly toxic to herbivores. In many cases the presence of an endophyte appears to benefit the plant by increasing drought resistance, or by increasing resistance to attack by insects, thus improving the overall survivability of the grass. This review will focus on alkaloids that have been reported in endophyte-infected grasses.

Acremonium↗

Molecular biology and evolution of the grass endophytes.

Acremonium coenophialum Morgan-Jones et W. Gams is a maternally transmitted fungal symbiont (endophyte) of the important forage grass Festuca arundinacea Schreb. (tall fescue), and provides biological protection and enhanced fitness to its host, but its anti-mammalian ergot alkaloids detract from the usefulness of tall fescue as forage for livestock. Molecular genetic techniques and materials are being developed in order to specifically eliminate the gene(s) encoding the first enzyme in ergot alkaloid biosynthesis. These techniques will also facilitate basic studies, such as host-fungus compatibility or biosynthesis of insecticidal alkaloids. Molecular phylogenetics indicate that endophytes related to A. coenophialum have evolved on multiple occasions from strains of Epichloë typhina (Ascomycotina, Clavicipitaceae), for which the sexual cycle is known. These studies also reveal significant diversity among seedborne endophytes in individual grass species. Thus, the endophytes are an important source of biochemical potential and genetic diversity in grass-fungus symbiota.

Acremonium↗

Does an asexual endophyte symbiont alter life stage and long-term survival in a perennial host grass?

Asexual, seedborne endophytic fungi in perennial grasses are often viewed as strong mutualists because fitness of the symbiont and host grass are closely coupled. However, at least for some native grasses, the asexual endophyte, Neotyphodium, acts parasitically, yet remains at high frequencies in natural populations. Most previous studies of Neotyphodium effects on host survival have been short term relative to the long life span of the perennial grass host. We therefore tested the hypothesis that Neotyphodium alters the survival in various life stages and long-term survival of adult native Arizona fescue (Festuca arizonica). To test the former, we planted 40 infected (E+) and 40 uninfected (E-, endophyte removed) seeds from four different maternal plants in the field under ambient conditions. We followed survival of seeds, seedlings, and adult plants over a 5-year period. To test the latter, we determined the infection of 1633 adult plants and followed their survival over the next 5-7 years. E+ seeds did not differ from uninfected seeds in terms of overall survival from seed germination to seedling to adult. However, the shape of the survival curve differed, with E+ plants showing higher mortality in early life stages. E+ adult plants did not differ from E- plants in long-term survival. Survival was generally very high during the study, which included a severe and prolonged drought. Infection by asexual Neotyphodium does not increase survival in early life stages or that of adult plants. Because asexual, vertically transmitted symbionts are predicted by evolutionary theory to be strong mutualists, the persistence of high infection frequencies in natural populations without long-term benefits to the host remains enigmatic. One possible explanation is that the long life span of the perennial host and low seedling recruitment may obscure either the costs or benefits of endophyte infection.

Festuca↗