Fungal Crop Research Articles

Can interaction specificity in the fungus-farming termite symbiosis be explained by nutritional requirements of the fungal crop?

Fungus-growing termites are associated with genus-specific fungal symbionts, which they acquire via horizontal transmission. Selection of specific symbionts may be explained by the provisioning of specific, optimal cultivar growth substrates by termite farmers. We tested whether differences in in vitro performance of Termitomyces cultivars from nests of three termite species on various substrates are correlated with the interaction specificity of their hosts. We performed single-factor growth assays (varying carbon sources), and a two-factor geometric framework experiment (simultaneously varying carbohydrate and protein availability). Although we did not find qualitative differences between Termitomyces strains in carbon-source use, there were quantitative differences, which we analysed using principal component analysis. This showed that growth of Termitomyces on different carbon sources was correlated with termite host genus, rather than host species, while growth on different ratios and concentrations of protein and carbohydrate was correlated with termite host species. Our findings corroborate the interaction specificity between fungus-growing termites and Termitomyces cultivars and indicate that specificity between termite hosts and fungi is reflected both nutritionally and physiologically. However, it remains to be demonstrated whether those differences contribute to selection of specific fungal cultivars by termites at the onset of colony foundation.

A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants.

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies have described the behaviors connected with disease defense, and the associated use of chemicals and antimicrobials, no common visual reference has been made. The main aim of this study was to record short clips of behaviors involved in disease defense, both prophylactically and directly targeted towards an antagonist of the colony following infection. To do so we used an infection experiment, with sub-colonies of the leaf-cutting ant species Acromyrmex echinatior, and the most significant known pathogenic threat to the ants' fungal crop (Leucoagaricus gongylophorus), a specialized pathogenic fungus in the genus Escovopsis. We filmed and compared infected and uninfected colonies, at both early and more advanced stages of infection. We quantified key defensive behaviors across treatments and show that the behavioral response to pathogen attack likely varies between different castes of worker ants, and between early and late detection of a threat. Based on these recordings we have made a library of behavioral clips, accompanied by definitions of the main individual defensive behaviors. We anticipate that such a guide can provide a common frame of reference for other researchers working in this field, to recognize and study these behaviors, and also provide greater scope for comparing different studies to ultimately help better understand the role these behaviors play in disease defense.

A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies have described the behaviors connected with disease defense, and the associated use of chemicals and antimicrobials, no common visual reference has been made. The main aim of this study was to record short clips of behaviors involved in disease defense, both prophylactically and directly targeted towards an antagonist of the colony following infection. To do so we used an infection experiment, with sub-colonies of the leaf-cutting ant species Acromyrmex echinatior, and the most significant known pathogenic threat to the ants' fungal crop (Leucoagaricus gongylophorus), a specialized pathogenic fungus in the genus Escovopsis. We filmed and compared infected and uninfected colonies, at both early and more advanced stages of infection. We quantified key defensive behaviors across treatments and show that the behavioral response to pathogen attack likely varies between different castes of worker ants, and between early and late detection of a threat. Based on these recordings we have made a library of behavioral clips, accompanied by definitions of the main individual defensive behaviors. We anticipate that such a guide can provide a common frame of reference for other researchers working in this field, to recognize and study these behaviors, and also provide greater scope for comparing different studies to ultimately help better understand the role these behaviors play in disease defense.

Disentangling nutritional pathways linking leafcutter ants and their co-evolved fungal symbionts using stable isotopes.

Leafcutter ants are the ultimate insect superorganisms, with up to millions of physiologically specialized workers cooperating to cut and transport vegetation and then convert it into compost used to cultivate co‐evolved fungi, domesticated over millions of years. We tested hypotheses about the nutrient‐processing dynamics governing this functional integration, tracing 15N‐ and 13C‐enriched substrates through colonies of the leafcutter ant Atta colombica. Our results highlight striking performance efficiencies, including rapid conversion (within 2 d) of harvested nutrients into edible fungal tissue (swollen hyphal tips called gongylidia) in the center of fungus gardens, while also highlighting that much of each colony's foraging effort resulted in substrate placed directly in the trash. We also find nutrient‐specific processing dynamics both within and across layers of the fungus garden, and in ant consumers. Larvae exhibited higher overall levels of 15N and 13C enrichment than adult workers, supporting that the majority of fungal productivity is allocated to colony growth. Foragers assimilated 13C‐labeled glucose during its ingestion, but required several days to metabolically process ingested 15N‐labeled ammonium nitrate. This processing timeline helps resolve a 40‐yr old hypothesis, that foragers (but apparently not gardeners or larvae) bypass their fungal crops to directly assimilate some of the nutrients they ingest outside the nest. Tracing these nutritional pathways with stable isotopes helps visualize how physiological integration within symbiotic networks gives rise to the ecologically dominant herbivory of leafcutter ants in habitats ranging from Argentina to the southern United States.

Dynamic environments of fungus-farming termite mounds exert growth-modulating effects on fungal crop parasites.

This study investigated for the first time the impact of the internal mound environment of fungus-growing termites on the growth of fungal crop parasites. Mounds of the termite Odontotermes obesus acted as (i) temperature and relative humidity (RH) 'stabilisers' showing dampened daily variation and (ii) 'extreme environments' exhibiting elevated RH and CO2 levels, compared to the outside. Yet, internal temperatures exhibited seasonal dynamics as did daily and seasonal CO2 levels. During in situ experiments under termite-excluded conditions within the mound, the growth of the crop parasite Pseudoxylaria was greater inside than outside the mound, i.e., Pseudoxylaria is 'termitariophilic'. Also, ex situ experiments on parasite isolates differing in growth rates and examined under controlled conditions in the absence of termites revealed a variable effect with fungal growth decreasing only under high CO2 and low temperature conditions, reflecting the in situ parasite growth fluctuations. In essence, the parasite appears to be adapted to survive in the termite mound. Thus the mound microclimate does not inhibit the parasite but the dynamic environmental conditions of the mound affect its growth to varying extents. These results shed light on the impact of animal-engineered structures on parasite ecology, independent of any direct role of animal engineers.

Diversity and dynamics of the DNA and cDNA-derived bacterial compost communities throughout the Agaricus bisporus mushroom cropping process

The cultivation of Agaricus bisporus involves the conversion of agricultural materials via fermentation into utilisable simple sugars as a nutrient source for the fungal crop during mushroom cropping. However, little is currently known about the role of the bacterial community contributing to the fermentation process. In this investigation we characterised the composition and dynamics of the DNA and cDNA-derived bacterial populations throughout a commercial mushroom cropping process using MiSeq sequencing. Both methods indicated substantial changes in the bacterial community structure after the first flush of the mushroom crop. However, differences were observed between the composition of the bacterial community determined by each of the two methods. The cDNA-derived community indicated that thermotolerant genera with known sulphur-reducing characteristics were highly active up to the first flush. Activity of the phyla Actinobacteria and Firmicutes was observed to increase as fermentation progressed, indicating that the members of these phyla played prominent roles in the conversion of wheat straw into utilisable sugars. The cDNA-derived community comprised genera with roles in the nitrification process that became highly active at post flush 1. Subsequent chemical analysis of extractable nitrate indicated that substantial nitrification occurred up until the termination of the cropping process. This study has demonstrated that a highly dynamic bacterial community is present throughout the mushroom cropping process.

Fungus-Farming Termites Selectively Bury Weedy Fungi that Smell Different from Crop Fungi.

Mutualistic associations such as the fungal farms of insects are prone to parasitism and are consequently vulnerable to attack by weeds and pests. Therefore, efficient farm management requires quick detection of weeds for their elimination. Furthermore, if the available weedicides are non-specific, then the ability of insects to discriminate between crop and weeds becomes essential for targeted application of such compounds. Here, we demonstrate for the first time in fungus-farming insects, that worker castes of the fungus-growing termite Odontotermes obesus discriminate between their crop (Termitomyces) and the weedy (Pseudoxylaria) fungi, even if exposed to only fungal scents. Termites respond to the presence of fungal mycelium or scent alone, by burying the weed with the offered material such as soil or agar, possibly anointing the weed with chemicals in the process. The scent profiles of crop and weedy fungi are distinct and the differences are likely exploited by termites to selectively mount their defences. Sesquiterpene compounds such as aristolene and viridiflorol, which are absent from crop odours, may constitute the "weedy scent". Our results provide a general mechanism of how other fungus-farming insects could avoid indiscriminate application of non-specific fungicides which could lead to poisoning their crops, and have bearing on the stability of the mutualism between termites and their crop fungus in the face of parasitism by weedy fungi.

Tryptorubin A: A Polycyclic Peptide from a Fungus-Derived Streptomycete.

Fungus-growing ants engage in complex symbiotic relationships with their fungal crop, specialized fungal pathogens, and bacteria that provide chemical defenses. In an effort to understand the evolutionary origins of this multilateral system, we investigated bacteria isolated from fungi. One bacterial strain (Streptomyces sp. CLI2509) from the bracket fungus Hymenochaete rubiginosa, produced an unusual peptide, tryptorubin A, which contains heteroaromatic links between side chains that give it a rigid polycyclic globular structure. The three-dimensional structure was determined by NMR and MS, including a 13C-13C COSY of isotopically enriched material, degradation, derivatives, and computer modeling. Whole genome sequencing identified a likely pair of biosynthetic genes responsible for tryptorubin A’s linear hexapeptide backbone. The genome also revealed the close relationship between CLI2509 and Streptomyces sp. SPB78, which was previously implicated in an insect–bacterium symbiosis.

Lysobacter PilR, the Regulator of Type IV Pilus Synthesis, Controls Antifungal Antibiotic Production via a Cyclic di-GMP Pathway.

Lysobacter enzymogenes is a ubiquitous soil gammaproteobacterium that produces a broad-spectrum antifungal antibiotic, known as heat-stable antifungal factor (HSAF). To increase HSAF production for use against fungal crop diseases, it is important to understand how HSAF synthesis is regulated. To gain insights into transcriptional regulation of the HSAF synthesis gene cluster, we generated a library with deletion mutations in the genes predicted to encode response regulators of the two-component signaling systems in L. enzymogenes strain OH11. By quantifying HSAF production levels in the 45 constructed mutants, we identified two strains that produced significantly smaller amounts of HSAF. One of the mutations affected a gene encoding a conserved bacterial response regulator, PilR, which is commonly associated with type IV pilus synthesis. We determined that L. enzymogenes PilR regulates pilus synthesis and twitching motility via a traditional pathway, by binding to the pilA promoter and upregulating pilA expression. Regulation of HSAF production by PilR was found to be independent of pilus formation. We discovered that the pilR mutant contained significantly higher intracellular levels of the second messenger cyclic di-GMP (c-di-GMP) and that this was the inhibitory signal for HSAF production. Therefore, the type IV pilus regulator PilR in L. enzymogenes activates twitching motility while downregulating antibiotic HSAF production by increasing intracellular c-di-GMP levels. This study identifies a new role of a common pilus regulator in proteobacteria and provides guidance for increasing antifungal antibiotic production in L. enzymogenesIMPORTANCE PilR is a widespread response regulator of the two-component system known for regulating type IV pilus synthesis in proteobacteria. Here we report that, in the soil bacterium Lysobacter enzymogenes, PilR regulates pilus synthesis and twitching motility, as expected. Unexpectedly, PilR was also found to control intracellular levels of the second messenger c-di-GMP, which in turn inhibits production of the antifungal antibiotic HSAF. The coordinated production of type IV pili and antifungal antibiotics has not been observed previously.

Phenylpyrroles: 30 Years, Two Molecules and (Nearly) No Resistance

Phenylpyrroles are chemical analogs of the natural antifungal compound pyrrolnitrin. Fenpiclonil, but mainly fludioxonil are registered against multiple fungal crop diseases since over 25 years for seed or foliar treatment. They have severe physiological impacts on the pathogen, including membrane hyperpolarization, changes in carbon metabolism and the accumulation of metabolites leading to hyphal swelling and burst. The selection and characterization of mutants resistant to phenylpyrroles have revealed that these fungicides activate the fungal osmotic signal transduction pathway through their perception by a typical fungal hybrid histidine kinase (HHK). The HHK is prone to point mutations that confer fungicide resistance and affect its sensor domain, composed of tandem repeats of HAMP motifs. Fludioxonil resistant mutants have been selected in many fungal species under laboratory conditions. Generally they present severe impacts on fitness parameters. Since only few cases of field resistance specific to phenylpyrroles have been reported one may suspect that the fitness penalty of phenylpyrrole resistance is the reason for the lack of field resistance.

Lactarius deliciosus Fr. soil extraradical mycelium correlates with stand fruitbody productivity and is increased by forest thinning

Lactarius deliciosus is a widely marketed non-wood forest product in Spain but little is known about silvicultural treatments that would enhance the production of this valued mushroom in the forests. Estimating a fungal crop by collecting sporocarps is expensive. Therefore we tested quantifying soil extraradical mycelium as a more efficient alternative to the inventory of sporocarps in undisturbed forest stands and immediately following a forest thinning.L. deliciosus soil mycelium was highly correlated with fruit body production in our study area for the year the soil was sampled and also on the following year, providing us with a promising tool to both model and forecast mushroom crops. Heavier thinning intensities increased the quantities of soil mycelium and sporocarp production in the autumn after it was applied.

Liquid foraging behaviour in leafcutting ants: the lunchbox hypothesis

Optimal foraging theory makes clear predictions about the benefits of maximizing energetic returns per unit of foraging effort. However, predictions become less clear when animals belong to symbioses that would be destabilized by such foraging decisions. For instance, leafcutter ants are dominant herbivores in Neotropical ecosystems that harvest fresh vegetation and convert it into compost used to cultivate specialized fungus for food. Individual foragers have long been assumed to supplement their fungal diets by harvesting liquid nectar outside the symbiosis, although this has not been demonstrated in the field, and would probably destabilize the fine-tuned farming systems. By dissecting liquid storage organs in foragers of four sympatric Panamanian leafcutter ant species we found that liquid foraging is not a general strategy in the field. Moreover, while over 40% of these foragers returned to their nests without leaf fragments, these unladen ants were not more likely to carry liquids. Instead, we found support for a newly formulated ‘lunchbox hypothesis’ because most workers exited nests for foraging trips with midguts full of liquids that were depleted (assimilated and transferred to hindguts) if workers returned with a leaf fragment in the field or transported a load in laboratory experiments. Thus, in contrast to the destabilizing effects of external nectar foraging, these results provide a novel mechanism promoting symbiotic stability, as fungi provide fuel for foragers to harvest more substrate for fungal crop production.

Diospyros cuneata Inhibition of Fusarium oxysporum: Aqueous Extract and its Encapsulation by Ionic Gelation

The application of plant extracts to control fungal crop pathogens is an ecological strategy that could potentially be useful in agriculture. Aqueous extracts of some species of the genus Diospyros spp have been tested against fungal pathogens of crops. Nevertheless, there is no information about the inhibitory effect of aqueous extracts of Diospyros cuneata on the micelial growth of Fusarium oxysporum, a pathogen responsible for “Panama disease” and “vanilla stem rot disease”. Therefore, the antifungal activity of aqueous extracts of Diospyros cuneata leaves collected during the dry and rainy seasons was tested in-vitro against spores of Fusarium oxysporum. Only the aqueous extract from leaves collected during the dry season had an inhibitory effect on the micelial growth of asexual spores (2.5% minimum inhibitory concentration). Phytochemical analysis showed that both aqueous extracts contained mainly flavonoids and tannins; the chromatographic profile showed a larger abundance of polar compounds in the dry season extract. Furthermore, the antifungal activity observed is probably correlated with the abundance of some secondary metabolites produced by water stress and dry season conditions. Thebioactivityof aqueous extracts of Diospyros cuneata leaves could be stored and released through encapsulation; an effective example of this was tested using alginate-inulin to prepare microbeads by ionic gelation.

Somatic incompatibility and genetic structure of fungal crops in sympatric Atta colombica and Acromyrmex echinatior leaf-cutting ants.

Obligate mutualistic symbioses rely on mechanisms that secure host-symbiont commitments to maximize host benefits and prevent symbiont cheating. Previous studies showed that somatic incompatibilities correlate with neutral-marker-based genetic distances between fungal symbionts of Panamanian Acromyrmex leaf-cutting ants, but the extent to which this relationship applies more generally remained unclear. Here we showed that genetic distances accurately predicted somatic incompatibility for Acromyrmex echinatior symbionts irrespective of whether neutral microsatellites or AFLP markers were used, but that such correlations were weaker or absent in sympatric Atta colombica colonies. Further analysis showed that the symbiont clades maintained by A. echinatior and A. colombica were likely to represent separate gene pools, so that neutral markers were unlikely to be similarly correlated with incompatibility loci that have experienced different selection regimes. We suggest that evolutionarily derived claustral colony founding by Atta queens may have removed selection for strong incompatibility in Atta fungi, as this condition makes the likelihood of symbiont swaps much lower than in Acromyrmex, where incipient nests stay open because queens have to forage until the first workers emerge.

Evolutionarily advanced ant farmers rear polyploid fungal crops.

Innovative evolutionary developments are often related to gene or genome duplications. The crop fungi of attine fungus‐growing ants are suspected to have enhanced genetic variation reminiscent of polyploidy, but this has never been quantified with cytological data and genetic markers. We estimated the number of nuclei per fungal cell for 42 symbionts reared by 14 species of Panamanian fungus‐growing ants. This showed that domesticated symbionts of higher attine ants are polykaryotic with 7–17 nuclei per cell, whereas nonspecialized crops of lower attines are dikaryotic similar to most free‐living basidiomycete fungi. We then investigated how putative higher genetic diversity is distributed across polykaryotic mycelia, using microsatellite loci and evaluating models assuming that all nuclei are either heterogeneously haploid or homogeneously polyploid. Genetic variation in the polykaryotic symbionts of the basal higher attine genera Trachymyrmex and Sericomyrmex was only slightly enhanced, but the evolutionarily derived crop fungi of Atta and Acromyrmex leaf‐cutting ants had much higher genetic variation. Our opposite ploidy models indicated that the symbionts of Trachymyrmex and Sericomyrmex are likely to be lowly and facultatively polyploid (just over two haplotypes on average), whereas Atta and Acromyrmex symbionts are highly and obligatorily polyploid (ca. 5–7 haplotypes on average). This stepwise transition appears analogous to ploidy variation in plants and fungi domesticated by humans and in fungi domesticated by termites and plants, where gene or genome duplications were typically associated with selection for higher productivity, but allopolyploid chimerism was incompatible with sexual reproduction.

Trichoderma saturnisporum, a new biological control agent.

Biocontrol agents (BCAs) could be a viable alternative to chemicals in the management of fungal crop diseases. Screening for potential biocontrol and plant growth promoter isolates from a soil in Cádiz (Spain) was conducted. Several isolates showed antagonism in in vitro tests to several plant pathogens. Two isolates of Trichoderma saturnisporum (Ascomycetes, Hypocreales) were identified by sequencing of the rDNA region. One isolate was selected for further in vivo plant growth promotion and biological control assays. Results indicate that substrate application of T. saturnisporum improved plant quality and showed biological control activity against Phytophthora capsici and Phytophthora parasitica (Peronosporales, Peronosporaceae). There are a few references to T. saturnisporum isolated from different media but not its ability to promote plant growth or biocontrol. This is the first report of T. saturnisporum as a seedling growth promoter and as biological control agent.

Farming of a defensive fungal mutualist by an attelabid weevil.

The mutualism between fungus-growing animals and fungi is a classic example of a complex interspecies association. A handful of insects, notably the well-recognized fungus-farming ants, termites and beetles, have developed advanced agriculture, which includes seeding new gardens with crop propagules, improving growth conditions and protecting and harvesting the fungal crop. More examples, which could be called 'proto-fungiculture', involve fewer adaptations, as exemplified by marine snails that farm intertidal fungi on marsh grass. Recent work has indicated that the solitary leaf-rolling weevil Euops chinensis (family Attelabidae) has a protofarming symbiosis with the mycangial fungus Penicillium herquei (family Trichocomaceae). In this study, we investigated how the weevils create cradles (leaf-rolls) for their offspring and protect the fungal garden. We describe new specialized structures and behaviors that E. chinensis females use for leaf-rolling and fungus inoculation. The fungus P. herquei produces the antibiotic (+)-scleroderolide in laboratory culture and in leaf-rolls, which can serve to inhibit microbial 'weeds' and pests, thus protecting the fungal garden against potential infection. The fungiculture of E. chinensis differs from other advanced insect fungiculture systems because female weevils do not continuously tend the inoculated microbe and do not depend nutritionally on the fungus. The defensive role of the cultivated fungus makes the attelabid weevils exceptional in 'proto-fungiculture' animals.

Study of adaptive and agronomic characters in lines of common wheat Omskaya 37 carrying 1RS.1BL and 7DL-7Ai translocations

Lines derived from spring bread wheat Omskaya 37 and possessing wheat-rye translocation 1RS.1BL have been studied and tested for resistance to fungal pathogens, drought, crop yield, and parameters characterizing ecological plasticity and stability. Three lines have been studied with the use of the C-banding technique. It is shown that these lines, in addition to wheat-rye translocation 1RS.1BL, possess wheat-wheatgrass translocation 7DL-7Ai, where a segment of chromosome 7Ai of Agropyron elongatum (=Thinopyrum elongatum; =Thinopyrum ponticum) is translocated to the long arm of wheat chromosome 7D. It is concluded that the complex resistance of Omskaya 37 and its promising lines is related to the influence of a cluster of genes located on segments of rye and Agropyron chromosomes involved in the respective wheat-alien trans-locations. Several promising lines are recommended for further use in breeding programs. One of the lines we consider here, Lutescens 242/97-2-10, is submitted to a state variety trial for registration as the spring wheat variety Omskaya 41.

Effects of Changes in Precipitation and Temperature on Select Agrophage Risk in Poland, 1965-2009

Agriculture is a branch of the economy seriously influenced by climate change, particularly in terms of the incidence and damage potential of crop agrophages. Precipitation and air temperature recorded over the last 44 years in five select locations in Poland were analyzed in order to determine whether and to what extent climate change had actually occurred. Monthly precipitation totals and mean air temperatures were analyzed in terms of seasons, taking into consideration the course of vegetation of both spring and winter crops and development of pests. The aim of the study was to determine how precipitation and air temperature change and to present a preliminary evaluation of their effects on the level of damages of economically important agrophages, for several decades covered by nationwide monitoring in Poland. On the basis of results of descriptive statistics it was found that the highest value of the coefficient of variation for precipitation over a period of 44 years was recorded in autumn, while for air temperature it was in winter. After the 44-year period had been divided into four 11-year periods, the last 11 years (1999-2009) showed that the mean value of air temperature in relation to the years 1988-98 (1.5oC) decreased only in winter, although the mean value was still above zero (0.9oC). In the other seasons in the years 1999-2009 it was the highest. In turn, mean precipitation levels for the last 11 years were highest only in winter. An increase in precipitation in the winter months could have been one of the factors contributing to a reduction of populations of wintering pest stages. Stable precipitation patterns and increasing temperatures in spring could have contributed to the development of e.g. the European corn borer, while it may have limited the intensity of fungal crop diseases.

Allergen of the Month—Fusarium

Fusarium is a large genus of filamentous fungi widely found in soil, marine or river environments, and on plants throughout the world. While most species are saprobes, there are numerous species which are pathogenic and can cause crop damage, and if ingested, human disease, and are the most important toxigenic crop fungi in the northern temperate regions.1,2 A survey of plant-derived foodstuffs revealed traces of several Fusarium trichothecene toxins in a large majority of cereal-based foods, one eighth of fruits and vegetables, and none in pseudo-cereals amaranth, quinoa, and buckwheat.