The effect of the fungicide phosphite on ectomycorrhizal fungi

In Western Australia, the fungicide phosphite is being applied to selected native plant communities in order to reduce the impact of the root and collar rot pathogen, Phytophthora cinnamomi. The effect of this fungicide on the growth and function of ectomycorrhizal (ECM) fungi and their mycorrhizas was unknown. Taking the hypothesis that phosphite has a deleterious effect on mycorrhizal fungi, this study explored potential detrimental effects of phosphite on early colonising ectomycorrhizal fungi. Ten isolates of Scleroderma and Pisolithus from Western Australia, isolated from a range of host plants. These isolates were partnered with Agonis flexuosa, Melaleuca scabra, Eucalyptus globulus, E. sieberi and four clonal lines of E. marginata (jarrah) in vitro. The isolates that formed a mantle, Hartig net and epidermal cell elongation characteristic of a successful symbiosis, were chosen for further studies on two contrasting E. marginata clonal lines, that were resistant or susceptible to P. cinnamomi. Foliar drenching with phosphite induced different responses in the two clonal lines when they were non-mycorrhizal. Phosphite decreased root production in the resistant clone, and increased the number of plantlets that produced roots in the susceptible clonal line. Generally, 3 g phosphite/L reduced the host response to mycorrhizal infection, and mycorrhizas reduced root responses to phosphite compared to those seen in non-mycorrhizal plants. To determine if phosphite could have a direct inhibitory effect on the hyphae of ECM fungi, three isolates of Laccaria, Scleroderma and Pisolithus were grown in pure culture, on media containing a range of phosphite and phosphate concentrations. The biomass of Laccaria generally decreased as phosphite concentration increased at low phosphate concentrations. As phosphate concentration increased, the biomass of each Laccaria isolate generally increased irrespective of phosphite concentration. In hyphae of the three isolates of Laccaria, the increasing concentrations of phosphate in the media resulted in significant accumulation of phosphate. In two isolates, external phosphite supply had no effect on phosphate uptake. Scleroderma and Pisolithus tolerated the same concentration of phosphite as phosphate, while Laccaria was more sensitive to phosphite. There was a significant difference in growth between Laccaria isolates, while there was less variation between isolates of Scleroderma and Pisolithus. Scleroderma was most sensitive with two isolates being killed by 40 mM and the third being killed by 100 mM phosphite, while 120 – 140 mM phosphite was fungicidal to Laccaria and Pisolithus isolates. In the glasshouse, non-mycorrhizal seedlings of E. marginata, E. globulus and A. flexuosa were sprayed to run-off with 0 to 10 g phosphite/L, and then planted into soil naturally infested with early colonising mycorrhizal species. Phosphite had no effect on the percentage of roots infected with mycorrhizal fungi. In another experiment, E. globulus seedlings ectomycorrhizal with Scleroderma, Pisolithus and Descolea were treated with 0 to 10 g phosphite/L and infection of new roots by ectomycorrhizal fungi was assessed. At the recommended rate (5 g phosphite/L), phosphite had no effect on ectomycorrhizal formation, while at 10 g/L phosphite decreased infection by Descolea by 15%. An in vitro study was undertaken on a clonal line of E. marginata to determine if the foliar application of 3 g phosphite/L had any effect on the ability of Scleroderma and Pisolithus spores to germinate and infect roots. There was no significant difference in the percentage of infected primary and lateral root tips in phosphite and control plants inoculated with Scleroderma or Pisolithus spores. To determine if the soluble and cell wall bound peroxidases and phenolics involved in host defence responses are affected by phosphite treatment of the host, a series of interactions with E. marginata, ECM fungi and P. cinnamomi were examined. Phosphite significantly reduced P. cinnamomi lesion length in all mycorrhizal and non-mycorrhizal treatments and altered static peroxidase activity and phenolic concentrations in the roots of all non-mycorrhizal plants. Phosphite did not induce changes in peroxidase activity or phenolic concentration in roots of the susceptible clone when in indirect contact with Pisolithus. However, there was a general increase in peroxidase activity and phenolic concentration in roots of the resistant clone in the presence of Pisolithus and P. cinnamomi. In contrast, phosphite decreased peroxidase activity in the susceptible clone in the presence of Scleroderma and had no effect on soluble or cell wall bound phenolics. Phosphite did not alter peroxidase activity or phenolic concentration in roots of the resistant clone challenged by P. cinnamomi in the presence of either Scleroderma or Pisolithus. In contrast, phosphite significantly increased peroxidase activity, and decreased soluble phenolic concentration in the roots of the susceptible clone in the presence of Pisolithus. A glasshouse trial examined the effect of foliar applied phosphite (3 g/L) on P. cinnamomi infection of roots of mycorrhizal E. marginata plants. Laccaria, Scleroderma and Pisolithus mycorrhiza were established with seedlings and a P. cinnamomi susceptible clonal line of E. marginata prior to phosphite treatment. P. cinnamomi zoospores were inoculated to the root zone 10 days after phosphite application. P. cinnamomi was recovered from 84% and 52% of the untreated seedlings and clonal plants respectively, whether they were ectomycorrhizal or not. By contrast, in phosphite treated plants, P. cinnamomi was recovered in 10% of seedlings and 6% of clonal plants. There was no difference in P. cinnamomi recovery between mycorrhizal types in seedlings and clonal plants. More P. cinnamomi was recovered from mycorrhizal than non-mycorrhizal clonal plants. There was no correlation between the extent of mycorrhizal fungal colonisation and the percentage of P. cinnamomi infected roots in clonal plants or seedlings. Overall conclusions Although only a few ECM fungi and host species were examined in this study, it appears that phosphite, when used at the recommended rate (5 g/L), may not have a detrimental effect on ECM formation. The concentration of phosphite that is fungicidal to ECM fungi in vitro is generally in excess of levels that would be found in treated plant tissues. However, when the recommended rate was exceeded it was shown that phosphite significantly decreased infection by Descolea. This study has shown that there is variation between genera of ECM fungi, host plants, type of plant (clonal material or seedlings) in response to phosphite. However, this study did not take into account differing phosphate concentrations and its effect on phosphite and mycorrhizal interactions, which would further increase these variations. This demonstrates that generalisations cannot be made on the effect of phosphite on ECM fungi and ECM plants.