**3. Utilization of excreta-derived nutrients by fungi**

Utilization of cormorant-derived N by fungi was demonstrated by investigating the natural 15N abundance in fruit bodies of litter- and wood-decomposing fungi collected in the study sites. 15N enrichments in plant tissues, forest floor materials, and mineral soils due to excreta deposition were demonstrated in the cormorant colonies at Isaki Peninsula (Section 1.3; Fig. 1), which was associated with such processes as trophic enrichment through aquatic food webs and ammonia volatilization from soils (Kameda et al., 2006). Using natural 15N abundance as a natural tracer thus makes it possible to test whether fungi utilized excretaderived N in the colonized forests.

The 15N values of fruiting bodies at Site C were 0.1 to 1.5‰ on average and at similar levels to that in precipitation at the vicinity of the study sites (Fig. 7) and were within the range for saprobic fungi previously reported (e.g., Kohzu et al., 1999; Trudell et al., 2004). 15N was significantly (generalized linear model, 2=39.0, P<0.001) different among Sites C, P, and A and was significantly (2=15.4, P<0.001) higher in litter- than in wood-decomposing fungi (Fig. 7). Mean 15N values of fruiting bodies were in the order: Sites A > P > C for both litterand wood-decomposing fungi (Fig. 7). 15N of dead needles, forest floor materials, and woody debris were also higher at Sites P and A than at Site C, and fruiting bodies of fungi

Excess Supply of Nutrients, Fungal Community, and Plant Litter

**4.1 Excreta addition reduced fungal growth** 

discussed above.

are shown. \* P<0.05.

Decomposition: A Case Study of Avian-Derived Excreta Deposition in Conifer Plantations 181

Linear growth rates of 22 fungal isolates (12 basidiomycetes, 11 ascomycetes, and 1 zygomycete) were compared between media C and P. Nineteen of the 22 isolates were collected in the study sites, and another three isolates in the Basidiomycota were obtained from a culture collection. The mean value of linear growth rates on medium P was significantly lower than that on medium C (Fig. 8), indicating that excreta of the cormorants generally suppressed the mycelial extension of fungi. When taxonomic groups of fungi were examined separately, the linear growth rates for the Basidiomycota were significantly (paired t-test, n=12, P<0.05) lower on medium P than on medium C, whereas the difference was not significant for the Ascomycota (paired t-test, n=11, P>0.05). These results are consistent with the field measurements showing that hyphal lengths in needle and twigs were shorter at Site P, where the forest floor suffered excreta deposition, than at Site C and that the reduction was obvious for clamp-bearing hyphae that belong to the Basidiomycota (Fig. 3). Possible inhibitory factors responsible for the decrease of fungal growth include the toxicity of ammonia and uric acid and the higher pH and salt concentration in excreta, as

C P

\*

Medium

Fig. 8. Linear growth rate of fungal colony on media C and P at 20°C under pure culture conditions. Medium P contained excreta. The original data are in Osono et al. (2006b). Values indicate means ± standard errors for 22 fungal species tested. Results of paired t-tests

Another pure culture decomposition test was carried out for 13 (eight basidiomycetes and five ascomycetes) of the 22 fungal isolates to evaluate the effect of excreta addition on decomposition (Osono et al., 2006b). Dead needles of *C. obtusa* collected at Site C were used as a substratum. The mean value of mass loss of needles on medium P was significantly lower than that on medium C (Fig. 9), indicating that excreta of the cormorants generally reduced the fungal decomposition. This reduction in decomposition was due to the suppression of decomposition of acid-unhydrolyzable residue (AUR) in needles, as the mass loss of AUR was significantly lower on medium P than on medium C (Fig. 9). In contrast, the mass loss of total carbohydrates was not significantly different between the media C and P (Fig. 9). The mass loss of N was significantly lower on medium P than on medium C (Fig. 9), indicating more accumulation of N in needles when fungi were incubated on medium P. 15N of needles decomposed by fungi on medium P (1.21±0.15‰, mean ± standard error, n=13) was significantly (paired t-test, P<0.001, n=13) higher than that on medium C

0

5

mm/day

**4.2 Excreta addition retarded fungal decomposition of needles** 

10

were generally enriched in 15N relative to their substrata collected at the same sites. Fruiting bodies of litter-decomposing fungi at Sites P and A and those of wood-decomposing fungi at Site A had similar or higher 15N values than that in excreta (Fig. 7).

Fig. 7. Nitrogen stable isotope ratios of fruiting bodies of litter- () and wood-decomposing fungi () (T. Osono, unpublished). Nitrogen stable isotope ratios of substrates for fungi are also shown: dead needles of *Chamaecyparis obtusa*; forest floor materials; woody debris. Values indicate means ± standard errors. Sites are as in Table 1. Horizontal lines indicate 15N values for excreta (means ± standard errors, n=12) and for precipitation (n=5) (Kameda et al., 2006). A total of 44 samples of fungal fruiting bodies representing 24 taxa were qualitatively collected from February 2000 to April 2003 and used for the analysis.

These results showed the effects of 15N-enriched excreta deposition on fruiting bodies of litter- and wood-decomposing fungi at the forest stands colonized by the cormorants. Previous studies have been successful in using N stable isotope ratios to demonstrate the transfer of animal-derived N to biotic components in terrestrial ecosystems, such as seabird rookeries (Mizutani and Wada, 1988; Wainright et al., 1998) and bear habitats where salmons are transferred from coastal waters to riparian forests (Wilkinson et al., 2005; Nagasaka et al., 2006). The uptake of excreta-derived N can alter metabolic activity of fungal mycelia, which is investigated in the next section.

#### **4. Reduction of fungal growth and decomposition by excreta**

The results of Sections 2 and 3 suggest possible effects of excreta on fungal growth and decomposition of plant tissues. These effects were verified with pure culture tests of fungal growth and decomposition on an agar medium supplemented with excreta in comparison with those on a control medium without excreta (Osono et al., 2006b).

In September 2000, water collectors with 15-cm diameter funnels on the top were installed on the forest floor within each of Sites C and P to collect throughfall (i.e., excess water shed from wet leaves onto the ground surface). The water samples from Site C contained throughfall (rainfall plus leaf leachates), whereas that from Site P contained the throughfall plus excreta of the cormorants. The water sample from Site P had higher pH and electrical conductivity and higher contents of total carbon, total N, and NH4-N than that from Site C (Osono et al., 2006b). Throughfall from Sites C and P was mixed with 2% agar (w/v) and sterilized to prepare agar media that were denoted here as media C and P, respectively.

#### **4.1 Excreta addition reduced fungal growth**

180 International Perspectives on Global Environmental Change

were generally enriched in 15N relative to their substrata collected at the same sites. Fruiting bodies of litter-decomposing fungi at Sites P and A and those of wood-decomposing fungi at

CPA

Precipitation = 0.2±1.5

Excreta = 13.2±0.4

Site

et al., 2006). A total of 44 samples of fungal fruiting bodies representing 24 taxa were qualitatively collected from February 2000 to April 2003 and used for the analysis.

**4. Reduction of fungal growth and decomposition by excreta** 

with those on a control medium without excreta (Osono et al., 2006b).

Fig. 7. Nitrogen stable isotope ratios of fruiting bodies of litter- () and wood-decomposing fungi () (T. Osono, unpublished). Nitrogen stable isotope ratios of substrates for fungi are also shown: dead needles of *Chamaecyparis obtusa*; forest floor materials; woody debris. Values indicate means ± standard errors. Sites are as in Table 1. Horizontal lines indicate 15N values for excreta (means ± standard errors, n=12) and for precipitation (n=5) (Kameda

These results showed the effects of 15N-enriched excreta deposition on fruiting bodies of litter- and wood-decomposing fungi at the forest stands colonized by the cormorants. Previous studies have been successful in using N stable isotope ratios to demonstrate the transfer of animal-derived N to biotic components in terrestrial ecosystems, such as seabird rookeries (Mizutani and Wada, 1988; Wainright et al., 1998) and bear habitats where salmons are transferred from coastal waters to riparian forests (Wilkinson et al., 2005; Nagasaka et al., 2006). The uptake of excreta-derived N can alter metabolic activity of fungal

The results of Sections 2 and 3 suggest possible effects of excreta on fungal growth and decomposition of plant tissues. These effects were verified with pure culture tests of fungal growth and decomposition on an agar medium supplemented with excreta in comparison

In September 2000, water collectors with 15-cm diameter funnels on the top were installed on the forest floor within each of Sites C and P to collect throughfall (i.e., excess water shed from wet leaves onto the ground surface). The water samples from Site C contained throughfall (rainfall plus leaf leachates), whereas that from Site P contained the throughfall plus excreta of the cormorants. The water sample from Site P had higher pH and electrical conductivity and higher contents of total carbon, total N, and NH4-N than that from Site C (Osono et al., 2006b). Throughfall from Sites C and P was mixed with 2% agar (w/v) and sterilized to prepare agar media that were denoted here as media C and P, respectively.

Site A had similar or higher 15N values than that in excreta (Fig. 7).

0

mycelia, which is investigated in the next section.

per mil

Linear growth rates of 22 fungal isolates (12 basidiomycetes, 11 ascomycetes, and 1 zygomycete) were compared between media C and P. Nineteen of the 22 isolates were collected in the study sites, and another three isolates in the Basidiomycota were obtained from a culture collection. The mean value of linear growth rates on medium P was significantly lower than that on medium C (Fig. 8), indicating that excreta of the cormorants generally suppressed the mycelial extension of fungi. When taxonomic groups of fungi were examined separately, the linear growth rates for the Basidiomycota were significantly (paired t-test, n=12, P<0.05) lower on medium P than on medium C, whereas the difference was not significant for the Ascomycota (paired t-test, n=11, P>0.05). These results are consistent with the field measurements showing that hyphal lengths in needle and twigs were shorter at Site P, where the forest floor suffered excreta deposition, than at Site C and that the reduction was obvious for clamp-bearing hyphae that belong to the Basidiomycota (Fig. 3). Possible inhibitory factors responsible for the decrease of fungal growth include the toxicity of ammonia and uric acid and the higher pH and salt concentration in excreta, as discussed above.

Fig. 8. Linear growth rate of fungal colony on media C and P at 20°C under pure culture conditions. Medium P contained excreta. The original data are in Osono et al. (2006b). Values indicate means ± standard errors for 22 fungal species tested. Results of paired t-tests are shown. \* P<0.05.

#### **4.2 Excreta addition retarded fungal decomposition of needles**

Another pure culture decomposition test was carried out for 13 (eight basidiomycetes and five ascomycetes) of the 22 fungal isolates to evaluate the effect of excreta addition on decomposition (Osono et al., 2006b). Dead needles of *C. obtusa* collected at Site C were used as a substratum. The mean value of mass loss of needles on medium P was significantly lower than that on medium C (Fig. 9), indicating that excreta of the cormorants generally reduced the fungal decomposition. This reduction in decomposition was due to the suppression of decomposition of acid-unhydrolyzable residue (AUR) in needles, as the mass loss of AUR was significantly lower on medium P than on medium C (Fig. 9). In contrast, the mass loss of total carbohydrates was not significantly different between the media C and P (Fig. 9). The mass loss of N was significantly lower on medium P than on medium C (Fig. 9), indicating more accumulation of N in needles when fungi were incubated on medium P. 15N of needles decomposed by fungi on medium P (1.21±0.15‰, mean ± standard error, n=13) was significantly (paired t-test, P<0.001, n=13) higher than that on medium C

Excess Supply of Nutrients, Fungal Community, and Plant Litter

examined in detail in the next section.

chemical constituents and nutrients.

Decomposition: A Case Study of Avian-Derived Excreta Deposition in Conifer Plantations 183

plant tissues in the field, because these fungi are primary agents removing recalcitrant compounds from the tissues and mobilizing nutrients (Osono, 2007). Consequently, it is hypothesized that the reduction in biomass (Fig. 3) and activity (Figs. 8 and 9) of ligninolytic basidiomycetes due to excreta addition would result in the reduction of long-term decomposition rates, the accumulation of recalcitrant compounds in decomposing plant tissues, and the concomitant immobilization of nutrients in the tissues. These hypotheses are

**5. Excreta deposition and decomposition of dead plant tissues in the field** 

estimate the decomposition processes in cormorant-colonized forests.

A litterbag experiment (Fig. 10) was performed to follow the two-year decomposition of needles and twigs of *C. obtusa* on the forest floor and to compare them between Sites C and P to estimate the possible effects of excreta on the decomposition (Osono et al., 2006a). In another field survey, mass and N content of coarse woody debris (CWD: logs, snags, and stumps with diameter equal to or greater than 10 cm) were examined in the study sites to

Fig. 10. Litterbags to study long-term decomposition of dead plant tissues in the field. In the

Over the two-year period, the mass loss was slower at Site P than at Site C and faster in needles than in twigs (Fig. 11). AUR mass loss in needles and twigs showed similar trends to mass loss of whole tissues and was slower at Site P than at Site C (Fig. 11). In contrast, mass loss of total carbohydrates in needles and twigs showed similar patterns between Sites C and P (data not shown; Osono et al., 2006a). These results support the hypotheses that the excreta deposition can lead to a reduction in decomposition rates and the accumulation of recalcitrant compounds in the decomposing plant tissues. The reduced AUR decomposition at Site P was primarily attributable to the reduced biomass and activity of ligninolytic

study of Osono et al. (2006a), needles and twigs collected at Site C were enclosed in polypropylene shade cloth (10 10 cm, mesh size of approx. 2 mm) and incubated on the forest floor at Sites C and P for two years. The litterbags were retrieved at 3- (the first year) or 6-month (the second year) intervals to analyze remaining mass and contents of organic

**5.1 Rate of mass loss of needles and twigs and recalcitrant compounds** 

basidiomycetes due to excess supply of excreta-derived N, as discussed above.

(0.51±0.06‰), suggesting that N in excreta was translocated into needles during the fungal decomposition on medium P.

When taxonomic groups of fungi were examined separately, the mean values of mass loss of AUR were significantly lower on medium P than on medium C for the Basidiomycota (paired t-test, n=8, P<0.05), whereas the difference was not significant for the Ascomycota (paired t-test, n=5, P>0.05), suggesting that AUR decomposition by basidiomycetes is more sensitive to excreta than that by ascomycetes. The AUR fraction, which has been commonly denoted as Klason lignin, contains lignin, tannin, and cutin (Preston et al., 1997) as well as humic substances produced secondarily during fungal decomposition (Fukasawa et al., 2009). The AUR fraction thus represents recalcitrant components in plant tissues and often limits decomposition and nutrient dynamics (Osono and Takeda, 2004). Because a high concentration of inorganic N can cause biochemical suppression of lignin-degrading enzymes responsible for AUR decomposition (Keyser et al., 1978; Fenn et al., 1981; Osono and Takeda, 2001), excreta rich in N are probably responsible for the observed sensitivity of ligninolytic basidiomycetes to excreta on medium P.

Fig. 9. Mass loss (% original mass) of dead needles of *Chamaecyparis obtusa* and of acidunhydrolyzable residue (AUR), total carbohydrates, and nitrogen in the needles on medium C and P. Medium P contained excreta. The original data are in Osono et al. (2006b). The needles were sterilized with ethylene oxide gas, inoculated with fungal isolates, and incubated at 20°C for 12 weeks in the dark. Values indicate means ± standard errors for 13 fungal species tested. Results of paired t-tests are shown. \* P<0.05, ns non significant.

In summary, the pure culture tests demonstrated that cormorant excreta negatively affected fungal growth and decomposition of needles and that ligninolytic basidiomycetes are more sensitive to excreta than ascomycetes. The reduced growth and decomposition by ligninolytic basidiomycetes due to excreta can alter the decomposition processes of dead

(0.51±0.06‰), suggesting that N in excreta was translocated into needles during the fungal

When taxonomic groups of fungi were examined separately, the mean values of mass loss of AUR were significantly lower on medium P than on medium C for the Basidiomycota (paired t-test, n=8, P<0.05), whereas the difference was not significant for the Ascomycota (paired t-test, n=5, P>0.05), suggesting that AUR decomposition by basidiomycetes is more sensitive to excreta than that by ascomycetes. The AUR fraction, which has been commonly denoted as Klason lignin, contains lignin, tannin, and cutin (Preston et al., 1997) as well as humic substances produced secondarily during fungal decomposition (Fukasawa et al., 2009). The AUR fraction thus represents recalcitrant components in plant tissues and often limits decomposition and nutrient dynamics (Osono and Takeda, 2004). Because a high concentration of inorganic N can cause biochemical suppression of lignin-degrading enzymes responsible for AUR decomposition (Keyser et al., 1978; Fenn et al., 1981; Osono and Takeda, 2001), excreta rich in N are probably responsible for the observed sensitivity of

0

10

ns \*

**Total carbohydrates Nitrogen**

0

5

C P

Medium

10

20

\* \*

**Needles AUR**

decomposition on medium P.

ligninolytic basidiomycetes to excreta on medium P.

0

0

10

20

30

C P

Medium

Fig. 9. Mass loss (% original mass) of dead needles of *Chamaecyparis obtusa* and of acidunhydrolyzable residue (AUR), total carbohydrates, and nitrogen in the needles on medium C and P. Medium P contained excreta. The original data are in Osono et al. (2006b). The needles were sterilized with ethylene oxide gas, inoculated with fungal isolates, and incubated at 20°C for 12 weeks in the dark. Values indicate means ± standard errors for 13 fungal species tested. Results of paired t-tests are shown. \* P<0.05, ns non significant.

In summary, the pure culture tests demonstrated that cormorant excreta negatively affected fungal growth and decomposition of needles and that ligninolytic basidiomycetes are more sensitive to excreta than ascomycetes. The reduced growth and decomposition by ligninolytic basidiomycetes due to excreta can alter the decomposition processes of dead

Mass loss (%)

10

20

plant tissues in the field, because these fungi are primary agents removing recalcitrant compounds from the tissues and mobilizing nutrients (Osono, 2007). Consequently, it is hypothesized that the reduction in biomass (Fig. 3) and activity (Figs. 8 and 9) of ligninolytic basidiomycetes due to excreta addition would result in the reduction of long-term decomposition rates, the accumulation of recalcitrant compounds in decomposing plant tissues, and the concomitant immobilization of nutrients in the tissues. These hypotheses are examined in detail in the next section.
