Modification of Susceptible and Toxic Herbs on Grassland Disease

Recent research shows that continuous overgrazing not only causes grassland biodiversity to decline, but also causes light fungal disease. Achnatherum inebrians is susceptible to fungal diseases and increases in prevalence during over grazing due its toxicity to livestock. This study aimed to examine the effects of A. inebrians on biological control organisms and levels of plant diseases in overgrazed grasslands in northwestern China. The results showed that A. inebrians plants were seriously infected by fungal diseases and that this led to a high incidence of the mycoparasitic species Ampelomyces quisqualis and Sphaerellopsis filum. In addition, the fungivore, Aleocharinae, was found only in the soil growing A. inebrians rather than in the overgrazed area without A. inebrians. Overall, in an overgrazed grassland fenced for one year, disease levels in blocks without A. inebrians were significantly higher than those in blocks with A. inebrians. Our findings indicated that the disease susceptible, toxic A. inebrians can help control plant disease levels in overgrazed grasslands.

Scientific RepoRts | 6:30635 | DOI: 10.1038/srep30635 (Supporting Information S1). In total, 14 plant species were listed in Table 1, while some species having very little proportion in the grassland or with no disease found were not listed. Eight of these listed species had significantly higher disease indexes in the fenced grassland compared with those in the overgrazed grassland (P < 0.05), with most of them being dominant or "common" species. However, there were two species of plants that had significantly lower disease indexes in the fenced grassland compared to the overgrazed grassland (P < 0.05) and there were two species showing no significant difference. Besides, two species were identified to be present only in overgrazed grassland, with indexes lower than 18.00. For plant species with a disease index higher than 18.00, eight species were found in the fenced grassland, while no species were found in the overgrazed grassland. Among these eight species, five are considered typical forage plants, including the dominant species, Elymus nutans which had a disease index around twice that observed in overgrazed grassland. Table 2, totally14 species were presented. Seven species having a significantly higher disease index in overgrazed grassland without A. inebrians than with A. inebrians present (P < 0.05). One species had no statistical significance in the two treatments whereas six species were diseased  Table 2. Disease indexes of plants in the overgrazed grassland without or with A. inebrians and fenced for one year (AO vs. AI). Note: 0.00 ± 0.00 indicates no disease was found; "AO" means the overgrazed grassland without A. inebrians and fenced for one year; "AI" means the overgrazed grassland with A. inebrians and fenced for one year; "P < 0.05" means the difference is significant.

Disease indexes of plants in the overgrazed grasslands without or with A. inebrians and fenced for one year (AO vs. AI). As shown in
only in overgrazed grassland without A. inebrians. There were nine species with indexes higher than 20.00 in the overgrazed grassland without A. inebrians, but none in the overgrazed grassland with A. inebrians. Among these nine species, six were predominant forage grasses. The disease index of the dominant species, E. nutans, in overgrazed grassland without A. inebrians was approximately three times of that in the overgrazed grassland with A. inebrians.

Disease indexes of plants in AO and in the overgrazed or fenced grasslands.
For comparing the disease indexes of plants in AO and in the overgrazed grassland, totally14 species were listed. All plants had higher disease indexes in AO than in the overgrazed grassland (P < 0.05) (Fig. 1a). For comparison, the disease indexes of plants in AO and in the fenced grassland, totally14 species were presented. There were ten plant species having higher disease indexes in AO than in the fenced grassland (P < 0.05). Three plant species had higher indexes in the fenced grassland than in AO (P < 0.05). One species had no significant difference. Eight of the nine forage plant species had higher indexes in AO than in the fenced grassland (P < 0.05). In general, the disease indexes were higher in AO than in the fenced grassland (P < 0.05) (Fig. 1b).

Disease indexes of plants in AI and in the overgrazed or fenced grasslands.
For comparing the disease indexes of plants in AI and in the overgrazed grassland, totally14 species were displayed. Compared with the disease indexes of plants in the overgrazed grassland, five forage plant species had significantly higher indexes (P < 0.05); two forage plants had no significant difference; and one forage plant had a lower index (P < 0.05). Six other unpalatable plant species had lower indexes (P < 0.05) in AI (Fig. 2a).
For comparing the disease indexes of plants in AI and in the fenced grassland, totally13 species were presented. Compared with the fenced grassland, seven plant species had significantly lower disease indexes (P < 0.05); three species had no significant difference; and three had significantly higher disease indexes (P < 0.05) in AI (Fig. 2b).  As identified, only one sub-family of arthropod, Aleocharinae (Staphylinidae), was fungivorous, and it was found only in the soil under tussock of A. inebrians, not in the overgrazed grassland without A. inebrians. The mean number of this insect was 2.12, 1.33 and 0.75 per block in Xiahe, Guinan and Alxa, respectively (not shown in Table).

Discussion
There are serious overgrazing problems in many grasslands of China 1-3 . It has been reported that the level of disease incidence is relatively lower in grasslands heavily grazed [6][7][8] . Similarly, our results show that most plant species, including dominant and common species, had lower disease incidence in grassland overgrazed for 18 years compared to grassland that had been fenced for the same period (regarded as a healthy grassland in this study) ( Table 1). This suggests that the levels of pathogenic fungi in overgrazed grassland is less than for fenced grassland. As such, mycophagous predators and mycoparasites of pathogens and other microorganisms maybe barely survive in overgrazed grassland leading to a subsequent lack of control for pathogenic species that can dramatically increase during pasture recovery (due to fencing).
Currently, fencing is a common way to help achieve grassland restoration 9,10 . As the overgrazed grassland is fenced or the grazing intensity is reduced for restoration, we can probably see more frequent plant disease occurrences in the future. Our study indicated that the disease indexes of forage plants were significantly higher in overgrazed grassland without A. inebrians and fenced for one year, compared with the grassland overgrazed for 18 years (P < 0.05) (Fig. 1a), and were also significantly higher than the disease indexes of forage plants in the grassland fenced for 18 years (P < 0.05) (Fig. 1b). On the contrary, when A. inebrians was present, the disease level of plants in the overgrazed grassland fenced for one year was significantly lower than the disease level of plants in the overgrazed grassland without A. inebrians and fenced for one year (P < 0.05) ( Table 2). A possible explanation is that seriously diseased A. inebrians provided enough pathogens to support the mycophagous predators and mycoparasites when pathogen was lacking in the slightly diseased grassland. Much research has shown that mycophagous predators and mycoparasites can reduce the prevalence of pathogens [19][20][21][22][23][24] . Accordingly, these biocontrol organisms could inhibit the explosion of pathogens by consuming them when overgrazed grassland is fenced for restoration.
In the overgrazed grassland with A. inebrians and fenced for one year, the results indicated that the forage plants had higher disease indexes, but the unpalatable plants had lower disease indexes, compared with those in the grassland overgrazed for 18 years (Fig. 2a).When compared with the grassland fenced for 18 years, on the whole, the disease indexes were significantly lower in the overgrazed grassland with A. inebrians and fenced for one year, but the disease levels of forage plant species were very close in the two treatments (Fig. 2b). These results indicate that disease levels are likely to increase in overgrazed grasslands after being fenced for restoration, but this outcome could be modified by diseased A. inebrians. Probably, it will gradually get to a similar level with the grassland fenced for 18 years treated as a healthy status. For a healthy grassland, there should be enough pathogens supporting mycophagous predators and mycoparasites. As a balance is achieved between them, the plant diseases can be kept at normal levels.
In contrast to the degraded grassland, A. inebrians can survive because of its toxicity to livestock that generally do not eat it 11,12 . Moreover, in contrast to a low disease incidence in overgrazed grassland, our results showed that A. inebrians were heavily infected by fungal diseases. These findings were consistent with the study by Li et al. 18 . We can see that the disease indexes of A. inebrians were significantly higher than other plants (as shown in Table 1). The resulted pathogens from A. inebrians provided a food source for their predators and parasites to live with. For example, the only mycophagous invertebrate, Aleocharinae, which consumes pathogens as food 25 was found only in the soil block under the tussock of A. inebrians, but not in overgrazed grassland without A. inebrians in Xiahe, Guinan and Alxa (not shown in Table). In addition, our study showed that a lot of A. quisqualis and S. filum parasitising powdery mildew and rust, respectively, were found on the leaves of A. inebrians. Ampelomyces quisqualis has been found to be parasitic on more than 65 species of powdery mildew 26,27 and used as biocontrol agent of powdery mildews (AQ10 Biofungicide; Ecogen Inc., Langhorne, PA, USA) 27 . Sphaerellopsis filum was reported to colonize 369 rust species 28 and decreased the severity of rust by reducing spore production [28][29][30][31] . These fungivorous insect and mycoparasites are therefore beneficial in the control of plant diseases in overgrazed grasslands when they are fenced for restoration.
This study showed that the degenerated grassland without A. inebrians was susceptible to diseases when restored by fencing. Past research has suggested that increased biodiversity could decrease disease levels 32-34 , namely, a popular hypothesis is called "diversity-disease". However, one review article indicated that even though biodiversity could generally reduce the prevalence of infectious diseases, the mechanism remained to be understood 35 . Our study indicates that the occurrence of plant diseases is probably determined by the balance in the pathogenic food chain. As a part of the food chain, pathogen, including fungi, bacteria and other microbes, not only can be a consumer but also can be consumed. When the food chain is broken, disease will have chances to breakout. Therefore, toxic and susceptible plants can be key species to control diseases in the grasslands.

Disease indexes of plants in grasslands fenced or overgrazed for 18 years (fenced vs. overgrazed grasslands).
This study compared the disease incidence in overgrazed or fenced grasslands in Xiahe county, i.e., Sangke grassland. The grazing rate in the overgrazed grassland was approximately 10 sheep per hectare. No livestock were grazed in the fenced grassland. The grasslands had been either overgrazed with livestock or fenced to exclude grazing for at least 18 years. We chose four blocks (10 m × 10 m) for each treatment. Using the five-point sampling method (the crossing point of two cross wires connecting 4 corners of a block and the 4 intermediate points between the center and each of the 4 corners), we collected plant samples and randomly removed 10 leaves or stems per plant species to record the disease rate and severity. The severity classification for each disease is presented in Supporting Information S8.
The formula for calculating the disease severity is specified as equations (1) Disease indexes of plants in overgrazed grassland without or with A. inebrians and fenced for one year (AO vs. AI). The same method mentioned above was applied to investigate plants disease status in AO and AI. These two treatments were fenced from January to late October of 2013 to examine the effects of seriously diseased A. inebrians on the disease status of grassland after fencing. Then the disease indexes of plants in AO and AI were compared with that in the grasslands fenced or overgrazed for 18 years, respectively. inebrians, all thesix sites were selected. In each site, the five-point sampling method was employed, and 10 leaves Scientific RepoRts | 6:30635 | DOI: 10.1038/srep30635 from each plant were used to record the disease rate and severity on A. inebrians. The presence of two mycoparasites on diseased leaves of A. inebrians was investigated in this study. One parasitized rust sori and the other was parasitic on powdery mildew.The mycoparasitism rate on rust sori was the percentage of rust sori that was colonised. The mycoparasitism rate on powdery mildew was the infection percentage, i.e., the area infected by the mycoparasite divided by the area infected by powdery mildew on the leaf.

Effects of A. inebrians on fungivorous arthropods.
Three sites were selected for this study, specifically, the grassland dominated by Elymus nutans in Xiahe, the wetland-type grassland dominated by Kobresia capillifolia in Guinan, and the arid grassland dominated by Pennisetum centrasiaticum in Alxa Left Banner. A. inebrians plants were found at these three sites. For each site, 30 soil blocks (0.4 m in diameter × 0.2 m in depth) were dug out, including 15 soil blocks for the overgrazed grassland without A. inebrians, and 15 under tussock of A. inebrians. These blocks were crushed to collect arthropods, which were brought back to the laboratory to identify whether they were fungivorous arthropods. Data analysis. Data were analyzed using SPSS 17.0 for Windows. The T-test for independent-samples was used to compare: 1) disease status of plants in the overgrazed or fenced grasslands; 2) disease status of plants in the overgrazed grasslands without or with A. inebrians after being fenced for one year (AO vs. AI); 3) disease status of plants in AO with that in the overgrazed or fenced grasslands; and 4) disease status of plants in AI with that in the overgrazed or fenced grasslands. Disease index of A. inebrians and mycoparasite rates were analyzed using one way ANOVA, and the number of mycophagous arthropods and dry weight of plant species were presented with means and standard errors (SE). The statistical significance was defined at the 95% confidence level (alpha = 0.05). All mean values were presented with ±1 SE.