Our data indicate that only ectomycorrhizal (ECM) fungi (and not wood rotting fungi) exhibited rapid responses to the application of diesel to a forest soil as evidenced by sequence and BLAST analysis of laccase gene copies. We hypothesize that laccase, being a single copy gene, is more difficult to detect relative to nrDNA gene copies which are part of a tandem repeat unit that can be repeated up to 10,000 times in a single genome. Hence, it is through positive growth responses to an added substrate that laccase genes reached detection levels in DNA extracts from forest these soils.
These results are in keeping with recent data that indicate that some ECM fungi (Gomphidius viscidus
and Laccaria bicolor
) can grow with diesel as their sole nutrient source [13
]. This is perhaps not surprising. ECM fungi are recently evolved from wood-rotting basidiomycetes [14
], and are obligate mutualists that form intimate associations with the roots of host trees. However, despite being dependent upon a mutualistic interaction with a host tree for carbon uptake and survival, many ECM fungi demonstrate the ability to utilize lignin as a nutrient source [15
]. Further, many produce laccases and other enzymes involved in carbon cycling in natural ecosystems [1
]. Indeed, recent evidence suggests that many EM fungi possess the ability to adopt at least a partial saprophytic habit in times of low host carbon availability [17
], or when carbon substrate is added [21
]. Because of these findings, it has been hypothesized that they may derive at least part of their carbon nutrition by enzymatic means [16
], though their capacity to break down wood is the topic of considerable debate [1
]. However, while they may not possess the capacity to fully break down the complex lignin polymers in wood, it has been hypothesized that ECM may play a significant role in carbon cycling via metabolism of the shorter chain phenolic breakdown products created by the metabolism of lignin by other microbes [20
]. These smaller compounds are analogous to components of many anthropogenic contaminants, represented here by diesel.
Our data revealed genera in ECM fungal 3 families that exhibited positive responses to diesel application; the Russulaceae (2 Russula
species), Atheliaceae (3 Piloderma
species), and the Trichoomataceae. Russuloid fungi are known to have laccase activity [23
], and species within the Russulaceae can exhibit significant oxidative enzyme activities [24
]. However, culture-based experiments involving measurement of potential remediation function of specific Russula
species obtained from non-contaminated soils indicate that these fungi are often poor performers [23
]. Despite this, it was noted that fungi are likely to perform differently in a natural setting and in symbiosis with their host trees than they do in pure culture, particularly if these fungi are obtained from contaminated soils which would presumably screen for more active isolates.
ECM fungi can often be classified by hyphal “exploration type”, which is based on their foraging distance in the soil, which can often be correlated with potential to produce oxidizing enzymes [25
species are classified by this system as a type that possesses high phenol oxidizing potential, and hence potentially have a high ability to utilize substrate via lignolysis. In keeping with this classification, the results of our study indicate that some Russula
species exhibit rapid, positive growth responses when exposed to diesel, supporting the hypothesis that at least some ECM fungi could fulfill a role in carbon cycling in forest ecosystems through the utilization of less recalcitrant phenolic compounds, if not from complete breakdown of woody substrate [20
The data also indicated positive responses by fungi in the family Tricholomataceae. However, genetic matches to members of the Tricholomataeae were low, 70% or less, indicating gaps in our knowledge regarding the diversity of these taxa. Indeed, molecular evidence indicates that this family is paraphyletic with subgroups that are comprised of several other families [26
]. Hence, conclusions regarding these taxa are less certain. Despite this, our studies of effects of decreased host photosynthetic potential on ECM communities indicate that both groups can be prevalent members of the soil hyphal community in pine forests [12
]. Further, Tricholoma
species possess laccase and proteinase activity [27
] and significant lignin-degrading potential has been demonstrated in several Tricholoma
]. Indeed, T. aurantium
may utilize low molecular weight lignin-derived substrates more efficiently than some traditional wood-rotting fungi [16
]. Though the laccase data indicate more species in this family than our ITS screen(most likely due to the paraphyletic nature of the Tricholomataceae) our data support the notion that members of this ECM genus could take part in carbon cycling in forest soils and could provide useful models for ectomycorrhiza-mediated restoration strategies in diesel contaminated soils.
Multiple laccase-like genes have been detected in Piloderma
(Atheliaceae) species [29
]. Because of this, and because they are often detected in organic soil layers, it has been hypothesized that they may have some role in lignin degradation processes [29
]. Our data indicate that Piloderma
taxa exhibit positive growth responses to diesel application, suggesting that they could be utilizing short chain phenolic compounds as nutrient sources. Unfortunately, over 3 fruiting seasons we found no Piloderma
fruiting bodies. Hence, identifications cannot be made to the species level at this time.
The majority of fungi in the soil pool did not exhibit a positive PCR response to diesel application. Most notable in this group of fungi are those in the Cortinariaceae and the Suilloid group (e.g., Suillus
species). Suilloid fungi tend to be long distance exploration types that in general lack lignin-degrading abilities [25
]. However, some Suillus
species can exhibit significant phenol-oxidation activity in field settings in which all factors that influence function are present and intact [12
] and in some remediation settings [23
sp, however, exhibit little if any remediation potential [23
], a finding that would seem to be supported by the lack of growth response we measured in our study. Cortinarioid fungi tend to be medium distance types, also generally lacking in phenol-oxidizing activity. Thus, in addition to being an indicator of physiological potential, Agerer’s exploration type may provide a rough indicator of fungi for potential screening for ectomycorrhizal-mediated phytoremediation of phenolic-based contaminants and as enzyme sources for industrial use.
In conclusion, our data indicate that the ECM community can respond rapidly to application of diesel as a potential nutrient source. Further, our results provide new information as to the range of environmental conditions that can be tolerated by some Russuloid and Athelioid fungi, and fungi in the Tricholomataceae. Finally, even if the ECM fungi in this system are playing no role in diesel degradation, ECM fungi can protect the host from damage when growing in harsh environments [30
] thereby enhancing their survivability in extreme habitats. Thus, our data provide new ECM models for further studies into phytoremediation or habitat restoration strategies that utilize specific host/fungal combinations.