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Neotyphodium and the Grass Endophytes

Introduction

Epichloe and Neotyphodium are an important group of clavicipitaceous fungi that form endophytic symbioses with temperate grasses in the Pooideae subfamily. Epichloe are sexual species with a heterothallic mating system. Neotyphodium are asexual fungi derived from Epichloe that form a symptomless infection. Epichloe form external stromata and thus have the potential to be transmitted horizontally. However, both are probably transmitted vertically in seed of colonsed plants. The fungi colonise intercellular spaces of leaf primordia, leaf sheaths and the vegetative tissues of the inflorescence. The fungi are associated with disorders of mammalian and insectivorous herbivores and thus have received considerable attentian. N. coenophialum in fescue and N. lolii in ryegrass have been intensely studied. This section will examine the mechanisms underlying the disorders of herbivores and the effects of the endophyte on plants.

Many grasses have associations with Balansioid fungi, the most commonly studied associations are between C3 grasses (cool season growth) and Neotyphodium. C4 grasses appear to have epibiotic associations more commonly. LINK

Infected Grasses

Many grasses (family Poaceae) in the subfamily Pooideae are associated with intercellular endophytic fungi. The fungi are located in leaves, leaf bases, rhizomes and seeds. The association has no obvious external signs and disease is not evident.

The association between Neotyphodium and C3 grasses is highly specific. Fungi isolated from one plant species, or even cultivar are unlikely to persist in a second cultivar following inoculation into the second. We know little about the mechanism underlying the specificity.

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Life Cycle of theFungus

Neotyphodium colonises the intercellular leaf and leaf base tissue. Usually the fungi can be observed as wavy hyphae located immediately below the epidermis. The fungus grows along the flowering stem as it elongates, and infects seeds as they form and develop. The fungus is transferred asexually from plant generation to plant generation within the seed in what is called 'vertical transmission'. The seed germinates and the fungi colonise the elongating leaf tissues. In other words, the fungus is clonal and the plant host sexual. Bear in mind that the host plant is sustained in agricultural settings, meaning the host is also effectively invariant despite sexual reproduction. The fungi have not been found outside the association. The fungi have no known sexual system and no other known mechanism to infect seedlings in the wild.

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Mechanisms of Activity

A. Feeding Deterrence

Defence chemicals are produced by the fungus, or the host when infected by the fungus. The chemicals disrupt several metabolic processes in herbivores that eat the grasses. The active metabolites are found in at least four broad categories:

  1. The ergopeptides ergovaline and ergovalinine are based on lysergic acid amide. Generally known as ergot alkaloids, they induce various metabolic disorders that reduce reproductive rates and growth of grazing animals. The signs include elevated temperature, vasoconstriction, and reduced prolactin in blood. Serum prolactin is a growth hormone that regulates feed intake in mammals. The ergot alkaloids also influence animal gait. The compounds are found in many other members of Balansioid fungi.
  2. Tremogenic neurotoxins Lolitrem A and B are indole diterpenoids. Along with paxilline alkaloids they induce various stiff legged gaits in sheep grazing on infected grass. In addition, they are toxic to or deter feeding by various insects.
  3. The unusual Pyrrolopyrazine Peramine is thought to influence insect activity. It is widely found to deter insect feeding. While peramine is the only compound so far chemically characterised, related lipophilic compounds have been indicated in some other endophyte associations.
  4. Pyrollizidine-based loline alkaloids reduce feeding by some insects, and acts in concert with the other compounds to reduce feeding and reproductive rates of many more insects. They also appear to be a factor in the allelopathic properties of infected fescue.

The chemicals influence livestock, insects, mites,and pathogenic nematodes. Other compounds are believed to influence fungal pathogens. The chemicals are systemically distributed in the plant; while the fungus colonises only the shoot, the deterrents influence shoot and root feeders.

Livestock have reduced rates of growth in cases where endophytic grass is the sole or major source of plant being eaten. The animals tend to develop unsteady gait and stand in water on hot days. The rate of milk production in dairy cattle is measurably reduced.

Insect attack is reduced in three ways in endophyte infected grasses. Fewer insects land on endophyte infected grasses. Insects will feed for shorter periods and less frequently on infected grasses even when they land and feed. Finally, rates of insect reproduction, specifically number of eggs laid and the viability of eggs, is reduced. Under field conditions, specific populations of fungi and insects have developed. As a consequence, the toxins produced by the endophyte deplete the insect population, but not completely. The endophyte/grass symbioticum is commonly not adapted to newly arrived insect species. With the arrival of new insects, grazing pressure is quite high until fungal genotypes able to deter the new herbivore become more common in the pasture. Typically, a long established pasture contains a mixture of fungal genotypes. This diversity among the fungi indicate relatively high rates of fungal mutation and significant selection pressures in the grazing environment.

B. Drought Tolerance of Plants

Endophytic grasses survive under intermittent drought longer than uninfected grasses. The mechanism is unclear, though change in osmotica within the plant tissues following colonisation by the endophyte may be important in some cases. Changes in osmotica suggest that the mechanism is based on changes in hormonal balances, either directly due to the fungi, or indirectly by the fungus influencing plant hormonal balances.

C. Plant Competition

In pastures, Neotyphodium may colonise up to 90% of the grass plants present. Neotyphodium infected plants are more competitive in mixed pastures (eg clover/ryegrass) containing uninfected plants. Low levels of infection at sowing (4%) may change to more than 40% within a few years, regardless of whether the pastures are infested with insects, affected by drought or diseased. Established endophyte infected plants have more tillers, and produce more seeds than uninfected plants, regardless of the grazing pressures. The variation between endophyte infected grasses is greater than uninfected grasses and they therefore have a wider region of adaptation than uninfected grasses. The competitive interaction appears to favour colonised plants overall, but the mechanisms underlying the resultant population of plants and fungi are complex and interrelated.

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Cost of the Endophyte

Published measures suggest the metabolic cost of the endophyte is undetectable. However, the question of cost to the plant is difficult to clarify. Endophyte infection may increase phosphate concentrations in the shoot tissue which in turn influence rates of photosynthesis. Nitrogen use appears to be more efficient in some plant/fungus genotypes, though the metabolites are themselves nitrogen-based. The fungus almost certainly produces the deterrent compounds under direction of the host plant. Yet, genetic variation among the fungi increases phenotypic variation of the plant population.

The value of measures of metabolic costs are debatable. Measures in my lab suggest that the cost is variable and determined in part by the density of the fungus in tissue, the presence of a mycorrhizal fungus in the root system and the phosphate nutrition of the plant. In many respects the endophyte/grass association is similar to mycorrhizas, with the added input of chemicals that influence interaction with other organisms.

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Australian Grasses

The distribution of endophytic fungi in native grasses in Australia is unknown. Several species have names which suggest that they may house toxic fungi (Shivery grass, Tremor grass etc). Preliminary studies indicate that natural colonisation is unusual in the southern part of Australia.

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Applications to Agriculture and Turf Culture

Fungi can be selected for production of specific toxins, and selections can be genetically manipulated in culture using classical techniques. The goal of plant breeders producing pasture grass is to select fungi that have only marginal effect livestock but significantly reduce disease and the damage caused by pests. Turf farmers have no interest in grazing by mammals, though they too wish to reduce insect damage. So far, reinoculation of grasses with desirable fungi has proven to be a major stumbling block due to the specificity of the association.

In addition, Neotyphodium does not survive in seed in hot conditions. In agronomic systems, the fungi may die out if the seed is stored for longer than 6 months, much less if the temperature of storage exceeds 36oC for more than 6 hours. Many so called “endophyte” seed stocks lack the fungus because storage conditions are primitive and seed is often kept for long periods in hot conditions.

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Conclusion

Members of a specific genus of fungus release highly toxic metabolites when in association with their grass host. These metabolites have been studied because of their effect on grazing livestock and herbivorous insect pests. The consequence of endophytes, sensu stricto, is that they modify the capacity of the host to function in competitive interactions with other plants. This selective advantage enables increases in the proportion of colonised grasses in pastures though not necessarily native vegetation. It also places herbivores under selective pressure to evolve resistance to the toxins. The wide array of metabolites found in these fungi is typical of all the Ascomycetous fungi that have been intensively studied, suggesting that formation and excretion of metabolically active compounds is a widespread and common phenomenon in fungi, and that the metabolites improve the capacity of fungi to survive in association with heterotrophic organisms. The phenomenon may apply even where the association between plant and fungus is less specific.

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References

Bacon CW & White JF (2000) Microbial Endophytes, Marcel Dekker.

Bischoff JF & White JF (2005) In The Fungal Community (3rd edit) eds Dighton J, White JF and Oudemans P. Taylor & Francis.

Redlin SC & Carris LM (1995) Endophytic Fungi in Grasses and Woody Plants. APS Press. Chs 7 and 8.

Ruders JA & Clay K (2005) In The Fungal Community (3rd edit) eds Dighton J, White JF and Oudemans P. Taylor & Francis.

Schardl CL & Leuchtmann A (2005) In The Fungal Community (3rd edit) eds Dighton J, White JF and Oudemans P. Taylor & Francis.

Scott B, Young CA, Tanaka A, & Parker EJ. (2007) Molecular and genetic analysis of symbiosis expressed secondary metabolites genes from the mutualistic endophytes Neotyphodium lolii and Epichloe festucae. In: Exploitation fo Fungi. Eds: Robson GD, van West P, Gadd GM. CUP.

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