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Maestre and colleagues collected data using a standardized field survey at 98 sites across 25 countries and 6 continents, fitted linear mixed models to data from all sites and grazing pressure levels, and then applied a multimodel inference procedure to select the set of best-fitting models. The authors found interactions between grazing and biodiversity in almost half of the best-fitting models, where increasing grazing pressure had positive effects on ecosystem services in colder sites with high plant species richness. However, increases in grazing pressure at warmer sites with high rainfall seasonality and low plant species richness interacted with soil properties to either increase or reduce the delivery of multiple ecosystem services. The authors’ findings highlight how increasing herbivore richness could enhance ecosystem service delivery across contrasting environmental and biodiversity conditions, enhancing soil carbon storage and reducing the negative impacts of increased grazing pressure. More

Trichoderma species collectionEighty strains were obtained from 100 soil samples collected from Zoige alpine wetland ecological regions in China. Details of the strains isolated from soil samples are given in Table 1. All strains were subsequently used for morphological identification, while fifty-seven were used for phylogenetic analysis.Table 1 Details of 80 Trichoderma isolates from the Zoige alpine wetland in this study.Full size tablePhylogenetic analysisThe ITS region used preliminarily as a species identification criterion was applied to TrichOKey at www.ISTH.info70. However, the ITS region has a low number of variable sites and long insertions in certain species; thus, it is unsuitable for a phylogenetic reconstruction of this group41. Our study successfully amplified most fragments of the genes tef1, rpb2, and acl1. We also designed a pair of new primers based on the full-length tef1 gene, 5′-GAGAAGTTCGAGAAGGTGAGC-3′ and 5′-ATGTCACGGACGGCGAAAC-3′, with which a 1.4-kb fragment was amplified for most isolates.All samples analyzed in our study were divided into 4 primary clades based on the gpd gene region, including 49 strains from the T. harzianum complex, 3 T. rossicum strains, 1 T. polysporum strain and one unknown species (4 Trichoderma sp. strains) (Fig. 1). Maximum parsimony analysis was conducted among 101 strains, with Protocrea farinosa (CPK 2472) and P. pallida (CBS 299.78) used as outgroup (Table 2). The dataset for the rpb2, tef1 and acl1 genes contained 3403 characteristics, among which 1152 were parsimony-informative, 988 were variable and parsimony-uninformative, and 1263 were constant. The most parsimonious trees are shown in Fig. 2 (tree length = 5054, consistency index = 0.6005, homoplasy index = 0.3995, retention index = 0.8105, rescaled consistency index = 0.4867).Figure 1Neighbor-joining tree based on partial gpd gene sequences from 57 Trichoderma isolates. Parsimony bootstrap values of more than 50% are shown at nodes.Full size imageTable 2 Trichoderma strain included in the multi-gene sequence analysis, with details of clade, strain number, location, and GenBank accessions of the sequences generated.Full size tableFigure 2Maximum parsimony tree of Trichoderma species inferred from the combined rpb2, tef1 and acl1 partial sequences. Maximum parsimony bootstrap values above 50% are shown at nodes. The tree was rooted with Protocrea farinose and P. pallida Isolates from this study are shown in red (new species in bold).Full size imageThe phylogram showed that 57 stains belonged to the following four clades: Harzianum, Polysporum, Stromaticum, and Longibrachiatum. The strains of the first three clades with neighboring named species were well supported by bootstrap values greater than 90%. The Harzianum clade contained T. alni, T. atrobrunneum, T. harzianum and T. pyramidale of the Trichoderma species complex. The Polysporum clade contained only T. polysporum, and the Stromaticum clade contained T. rossicum. The Longibrachiatum clade contained four strains of Trichoderma sp., T25, T43, T44 and T48, which were separated from any other known taxa of this clade showed a low bootstrap value (MPBP = 62%) with T. citrinoviride and T. saturnisporum. We thus regarded it as a new species and named it Trichoderma zoigense, as described in the next section.Growth ratesAs shown in Fig. 3, the genus Trichoderma from Zoige alpine wetland ecological regions was able to grow in a range from 15 to 35 °C, and the suitable growth temperature for most species ranged from 20 to 30 °C. All seven species identified had normal viability at relatively low temperature (15 °C), and they rarely grew well over 35 °C except for T. zoigense. For T. atrobrunneum, T. harzianum and T. pyramidale, the optimum growth temperature on CMD was 25 to 30 °C. T. alni and T. rossicum preferred a cool growth environment, with an optimum temperature of 25 °C, whereas T. zoigense was more partial to a hot environment, with an optimum temperature of 30 °C, and it even grew well up to 35 °C. T. polysporum was the only slow-growing species that grew with less than 6.0 mm/day between 15 and 30 °C and did not survive at 35 °C. The above results showed that all species had different growth rates but were not completely differentiated from each other on CMD. These species were roughly divided into four groups based on their optimum growth temperature.Figure 3Growth rates of 7 species of Trichoderma on CMD given as mm per day at five temperatures. The values were the means of 3–5 experiments, with 1–3 representative isolates per species.Full size imageRelationship with ecological factorsOur results revealed a substantial disparity in the number and distribution of Trichoderma species among Zoige alpine wetland ecological regions (Tables 3, 4). Table 3 showed that T. harzianum was found in all four soil types, but most isolates of this species were obtained from peat soil. T. rossicum, T. alni and T. zoigense were also present in meadow soil and subalpine meadow soil, whereas T. atrobrunneum was found in aeolian sandy soil and peat soil. T. polysporum was found only in peat soil.Table 3 Isolation frequency of Trichoderma species in different soil types (%).Full size tableTable 4 Isolation frequency of Trichoderma species in different soil layers (%) species.Full size tableIn regard to the different soil layers shown in Table 4, T. harzianum was widely distributed in the five soil layers at depths of 0–100 cm. T. rossicum, T. alni and T. zoigense were isolated mainly from the soil layers at depths of 0–50 cm. Both T. atrobrunneum and T. pyramidale were isolated from depths of 0–10 cm, and T. polysporum was found only in the soil layers at depths of 50–100 cm.Regarding isolation frequency, T. harzianum was the most common of the seven species with a 23% isolation frequency, and it was therefore the dominant species in the zone, while the rare species T. polysporum and T. pyramidale had the lowest isolation frequencies at 1%.TaxonomyNew speciesTrichoderma zoigense G.S. Gong & G.T. Tang, sp. nov. (Fig. 4).Figure 4Cultures and asexual morph of Trichoderma zoigense. (a–d). Cultures at 20 °C [(a) on CMD, 7 days; (b) on MEA, 4 days; (c) on PDA, 4 days; and (d) on SNA, 7 days]. (e) Conidiation tuft (CMD, 4 days). (f–k) Conidiophores and phialides (CMD, 5–7 days). (l) Chlamydospores (PDA, 8 days). (m) Conidia (CMD, 5 days). Scale bars: (e) = 2 mm; (f–m) = 10 μm.Full size imageMycoBank: MB 82114.Typification: CHINA. SICHUAN PROVINCE: Zoige Alpine Wetland, on soil, 29 June 2013, G.S. Gong T44 (holotype CGMCC3.20145). GenBank: ITS = KX632531; TEF = KX632588; RPB2 = KX632645; ACL1 = KX632702; GPD = KX632759.Etymology: zoigense (Latin), the specific epithet about the place where the type was found.Description: Cultures and anamorph: optimal growth at 25 °C on all four media. On CMD after 72 h, growth is 25–28 mm at 20 °C and 28–31 mm at 25 °C. Colony is dense and has a wavy to crenate margin. Surface becomes distinctly zonate and white to grayish-green but celadon to atrovirens later, and it is granular in the center and distinctly radially downy outside and shows whitish surface hyphae and reverse-diffusing croci to pale brown pigment (Fig. 4a). Aerial hyphae are numerous to punctate and long, forming radial strands, with white mycelial patches appearing in aged cultures (Fig. 4e). Autolytic excretions are rare, with no coilings observed. Conidiation was noted after 3–4 d at 25 °C, a yellow or greenish color appears after 7 days, conidiation is effuse, and in intense tufts, erect conidiophores occur around the plug and on aerial hyphae. They are mainly concentrated along the colony center, show a white color that turns green, and then finally degenerate, with conidia often adhering in chains. Conidiophores are short and simple with asymmetric branches. Branches produce phialides directly. Phialides are generally solitary along main axes and side branches and sometimes paired in the terminal position of the main axes, sometimes in whorls of 2–3. Phialides are 4.5–10.5 × 2–5 μm ((overline{x }) = 7.5 ± 1.5 × 3 ± 0.5, n = 50) and 1.5–2.5 μm ((overline{x }) = 2 ± 0.2) wide at the base, lageniform or ampulliform, mostly uncinate or slightly curved, less straight, and often distinctly widened in the middle (Fig. 4f–k). Conidia are 3–4.5 × 2.3–4 μm ((overline{x }) = 3.5 ± 0.3 × 3 ± 0.3, n = 50) and initially hyaline, and they turn green and are oblong or ellipsoidal, almost with constricted sides, and smooth, eguttulate or with minute guttules, with indistinct scars (Fig. 4m).On PDA, after 72 h, growth is 35–41 mm at 20 °C and 50–55 mm at 25 °C; and mycelium covers the plate after 5 days at 25 °C. Colonies are dense with wavy to crenate margins; and mycelia are conspicuously differentiated in width of the primary and secondary hyphae. Surface becomes distinctly zonate, yellowish-green to prasinous in color and celadon to atrovirens later, and it is farinose to granular in the center, distinctly radially downy outside, with whitish of surface hyphae and reverse-diffusing brilliant yellow to fruit-green pigment (Fig. 4c). Aerial hyphae are numerous, long and ascend several millimeters, forming radial strands, with white mycelial patches appearing in aged cultures. Autolytic excretions are rare; and no coilings are observed. Odor is indistinct or fragrant. Chlamydospores examined after 7 days at 4.5–9 × 4.5–7.5 μm ((overline{x }) = 6 ± 1.1 × 6 ± 0.7, n = 50), and they are terminal, intercalary, globose or ellipsoidal, and smooth (Fig. 4l). Conidiation is noted after 3–4 days and yellow or greenish after 7 days. Conidiophores are short and simple with asymmetric branches; conidia are greenish, ellipsoidal, and smooth.On SNA, after 72 h, growth is 13–15 mm at 20 °C and, 16–21 mm at 25 °C; and mycelium covers the plate after 12–13 days at 25 °C. Colony is similar to that on CMD, with a little wave margin, although mycelia are looser and slower on the agar surface. Aerial hyphae are relatively inconspicuous and long along the colony margin. Autolytic activity and coiling are absent or inconspicuous. No diffusing pigment or distinct odor are produced (Fig. 4d). Conidiation was noted after 3–4 days at 25 °C, and many amorphous, loose white or aqua cottony tufts occur, mostly median from the plug outwards, and they are confluent to masses up and white but then turn green. After 4–5 days, conidiation becomes dense within the tufts, which are loose at their white margins with long, straight, or slightly sinuous sterile ends in the periphery. Tufts consisting of a loose reticulum with branches often at right angles, give rise to several main axes. Main axes are regular and tree-like, with few or many paired or unpaired side branches. Branches are flexuous, and phialides are solitary along the main axes and side branches, and they are sometimes paired in the terminal position of the main axes, sometimes in whorls of 2–3 that are often cruciform or in pseudo-whorls up to 4. Phialides and conidia are similar to that on CMD.New records for ChinaTrichoderma atrobrunneum F. B. Rocha et al., Mycologia 107: 571, 2015 (Fig. 5).Figure 5Cultures and asexual morph of Trichoderma atrobrunneum. (a–d) Cultures at 25 °C [(a) on CMD, 7 days; (b) on MEA, 4 days; (c) on PDA, 15 days; and (d) on SNA, 7 days]. (e) Conidiation tuft (SNA, 7 days). (f–i,k,l) Conidiophores and phialides (CMD, 5–7 days). (j) Conidia (CMD, 6 days). (m) Chlamydospores (PDA, 7 days). Scale bars: (e) = 2 mm; (f–m) = 10 μm.Full size imageSpecimen examined: CHINA. SICHUAN PROVINCE: Zoige Alpine Wetland, on soil, 29 June 2013, G.S. Gong T42 (holotype CGMCC.20167). GenBank: ITS = KX632514; TEF = KX632571; RPB2 = KX632628; ACL1 = KX632685; GPD = KX632742.Description: Cultures and anamorph: optimal growth at 25 °C on all media. On CMD, after 72 h, growth is 35–37 mm at 20 °C and 46–53 mm at 25 °C; mycelium covers the plate after 5–6 days at 25 °C. Colonies show distinct zonation. Mycelia are loose and thin; hyphae are narrow, sinuous and often form strands on the margin (Fig. 5a). Aerial hyphae are slight, forming a thin white to green downy fluffy or floccose mat. The light brown or brown pigment is observed, with no distinct odor noted. Conidiophores are pyramidal, often with opposing and somewhat widely spaced branches, with the main axis and each branch terminating in a cruciate, sometimes verticillate, whorl of up to four phialides. Phialides are ampulliform to lageniform and 4.9–7.6 × 2.2–3.0 μm ((overline{x }) = 6 ± 0.7 × 2.5 ± 0.2, n = 50) and 1.5–2.5 μm ((overline{x }) = 1.5 ± 0.3) wide at the base (Fig. 5f–i,k,l). Conidia are 2.5–4 × 2.5–3.5 μm ((overline{x }) = 3 ± 0.3 × 3 ± 0.2, n = 50), yellow to green, smooth, and circular to ellipsoidal (Fig. 5j).On PDA, after 72 h, growth is 41–43 mm at 20 °C and 50–55 mm at 25 °C; and mycelium covers the plate after 5–6 days at 25 °C. Colonies show indistinct zonation. Mycelia are dense, opaque, and thick; hyphae are wide, sinuous and often form strands on the margin (Fig. 5c). Margin is thick and defined. Aerial hyphae are abundant and form a thick green downy mat. Conidiation forms abundantly within 4 days in broad concentric rings. Chlamydospores examined after 7 days are 5–9 × 5.5–8.5 μm ((overline{x }) = 6.5 ± 0.9 × 6.5 ± 0.9, n = 30), globose when terminal, smooth, and intercalary (Fig. 5m).On SNA, after 72 h, growth is 33–35 mm at 20 °C and 38–40 mm at 25 °C; and mycelium covers the plate after 7–8 days at 25 °C. Colonies show distinct zonation. Mycelia are thin and yellow to green; hyphae are wide and sinuous, with indistinct strands on the margin (Fig. 5d). Margin is thin and ill-defined. Aerial hyphae are slight, forming a thin green downy fluff appearing in the colony (Fig. 5e). Diffusing pigment was observed in a ring, and no distinct odor was noted. Conidiation is similar to CMD.Accepted species previously reported in ChinaTrichoderma alni Jaklitsch, Mycologia 100: 799. 2008 (Fig. 6).Figure 6Cultures and asexual morph of Trichoderma alni. (a–d). Cultures after 7 days at 25 °C [(a) on CMD; (b) on MEA; (c) on PDA; and (d) on SNA]. € Coilings of aerial hyphae (PDA, 6 days). (f–j,l). Conidiophores and phialides (CMD, 5–7 days). (k) Conidiation tuft (PDA, 7 days). (m) Conidia (CMD, 6 days). (n,o) Chlamydospores (PDA, 7 days). Scale bars: (e–j,l–o) = 10 μm; (k) = 2 mm.Full size imageDescription: Cultures and anamorph: Optimum growth at 25 °C on all media; no growth at 35 °C. On CMD, after 72 h, growth of 34–36 mm at 20 °C and 50–51 mm at 25 °C; and mycelium covers the plate after 5–6 days at 25 °C. Colonies show distinct zonation. Mycelia are loose and thin; hyphae are narrow and sinuous and often form strands on the margin (Fig. 6a). Aerial hyphae are slight and form a thin white to green downy, fluffy or floccose mat. No diffusing pigment or distinct odor is noted. Conidiophores are hyaline and thick, with side branches on several levels at the base of the elongations that are mostly paired and in right angles with phialides in whorls of 3–5. Phialides are 5.5–11.5 × 2–3.5 μm ((overline{x }) = 8 ± 1.4 × 2.5 ± 0.4, n = 50) and 1.5–2.5 μm ((overline{x }) = 2 ± 0.4) wide at the base, often short and wide, and ampulliform (Fig. 6f–j,l). Conidia are 3–4 × 2.5–3.5 μm ((overline{x }) = 3.5 ± 0.2 × 3 ± 0.2, n = 50), dark green, smooth, and ellipsoidal (Fig. 6m).On PDA, after 72 h, growth is 33–35 mm at 20 °C and 41–43 mm at 25 °C; and mycelium covers the plate after 6–7 days at 25 °C. Colonies show indistinct zonation. Mycelia are dense, opaque, and thick; hyphae are wide, sinuous and often form strands on the margin (Fig. 6c). Margin is thin and ill defined. Aerial hyphae are slight, coiled (Fig. 6e), forming a thin white to green downy, fluffy or floccose mat (Fig. 6k). Chlamydospores examined after 7 days are 6–9.5 × 5–8 μm ((overline{x }) = 7.5 ± 0.9 × 7 ± 0.9, n = 30), globose to oval when terminal, and smooth, and few are intercalary (Fig. 6n,o).On SNA, after 72 h, growth is 18–19 mm at 20 °C and 28–32 mm at 25 °C; and mycelium covers the plate after 6–7 days at 25 °C. Colonies show distinct zonation. Mycelia are thin and yellow to green; hyphae are wide and sinuous and show indistinct strands on the margin (Fig. 6d). Margin is thin and ill-defined. Aerial hyphae are slight and form a thin white downy, fluffy, or floccose mat appearing in distal parts of the colony. No diffusing pigment or distinct odor was noted. Conidiation is similar to CMD.Trichoderma harzianum Rifai, Mycol. Pap. 116: 38, 1969 (Fig. 7).Figure 7Cultures and asexual morph of Trichoderma harzianum. (a–d) Cultures after 7 days at 20 °C [(a) on CMD; (b) on MEA; (c) on PDA; and (d) on SNA]. (e) Conidiation tuft (CMD, 7 days). (f–j) Conidiophores and phialides (CMD, 5–7 days). (k) Conidia (CMD, 5 days). (l,m) Chlamydospores (PDA, 7 days). Scale bars: (e) = 2 mm; (f–m) = 10 μm.Full size imageDescription: Cultures and anamorph: optimal growth at 25 °C on all media. On CMD, after 72 h, growth is 34–38 mm at 20 °C and 46–53 mm at 25 °C; mycelium covers the plate after 5–6 days at 25 °C. Colonies show distinct zonation. Mycelia are loose and thin; hyphae are narrow, sinuous, and often form strands on the margin (Fig. 7a). Aerial hyphae are abundant and radiating and form thick green downy, fluffy, or floccose mats (Fig. 7e). No diffusing pigment, but fragrant odor noted. Conidiophores are pyramidal with opposing branches, with each branch terminating in a cruciate whorl of up to four or five phialides. Phialides are frequently solitary or in a whorl of three or four. Phialides are ampulliform to lageniform and often constricted below the tip to form a narrow neck of 4.5–8 × 2–3.5 μm ((overline{x }) = 6 ± 0.8 × 2.5 ± 0.3, n = 50) and 1–2.5 μm ((overline{x }) = 2 ± 0.3) wide at the base (Fig. 7f–j). Conidia are subglobose to ovoid, 3–4.5 × 2.5–3.3 μm ((overline{x }) = 3.5 ± 0.3 × 3 ± 0.2, n = 50), laurel-green to bright green, smooth, and ellipsoidal (Fig. 7k).On PDA, after 72 h, growth is 41–43 mm at 20 °C and 50–55 mm at 25 °C; and mycelium covers the plate after 5–6 days at 25 °C. Colonies show distinct zonation. Mycelia are dense, opaque, and thick; hyphae are wide and sinuous and often form strands on the margin (Fig. 7c). Margin is thick and ill defined. Aerial hyphae are abundant and radiating and form thick green downy, fluffy or floccose mats. Chlamydospores examined after 7 days are 5.5–9 × 5.5–9.0 μm ((overline{mathrm{x} }) = 7 ± 0.8 × 7 ± 0.8, n = 30), globose to oval when terminal and smooth, showing an almost unobserved intercalary (Fig. 7l,m).On SNA, after 72 h, growth is 33–35 mm at 20 °C and 38–40 mm at 25 °C; and mycelium covers the plate after 7–8 days at 25 °C. Colonies show distinct zonation. Mycelia are thin and green; hyphae are narrow and sinuous and show indistinct strands on the margin (Fig. 7d). Margin is thin and ill defined. Aerial hyphae are slight and form a thick downy, fluffy, or floccose mat appearing in the colony. No diffusing pigment or distinct fragrant odor was noted. Conidiation was similar to CMD.Trichoderma polysporum Rifai, Mycol. Pap. 116: 18, 1969 (Fig. 8).Figure 8Cultures and asexual morph of Trichoderma polysporum. (a–d) Cultures at 20 °C [(a) on CMD, 7 days; (b) on MEA, 15 days; (c) on PDA, 15 days; and (d) on SNA, 15 days]. (i) Conidiation tuft (PDA, 15 days). (e–h,j) Conidiophores and phialides (CMD, 5–7 days). (k) Chlamydospores (CMD, 7 days). (l) Conidia (PDA, 6 days). Scale bars: (i) = 2 mm; (e–h,j) = 10 μm.Full size imageDescription: Cultures and anamorph: optimal growth at 20 °C on all media, no growth at 35 °C. On CMD, after 72 h, growth is 14–16 mm at 20 °C and 9–12 mm at 25 °C; and mycelium covers the plate after 9–10 days at 20 °C. A colony is hyaline, thin and loose, with little mycelium on the agar surface, and it is indistinctly zonate but becomes zonate by conidiation in white tufts after 4–5 d and grass green to green after 6 days (Fig. 8a). Aerial hyphae are long and dense and forming little greenish aggregates that are granular to pulvinate. No pigment or odor. Conidiation noted after 4–5 days, and it is white to greenish, with sterile smooth to rough helical elongations in the distal zones from pustules. Conidiophores are hyaline and thick with side branches on several levels at the base of the elongations that are mostly paired and at right angles with phialides in whorls of 2–5. Phialides are 5–10.5 × 2.5–4 μm ((overline{x }) = 7 ± 1.9 × 3.5 ± 0.4, n = 50) and 2–4 μm ((overline{x }) = 3 ± 0.5) wide at the base, often short and wide and ampulliform (Fig. 8e–h,j). Conidia are 2.5–4 × 2–3 μm ((overline{x }) = 3.5 ± 0.4 × 2.5 ± 0.2, n = 50), hyaline, smooth, and ellipsoidal (Fig. 10l).On PDA, after 72 h, growth is 24–26 mm at 20 °C and 13–16 mm at 25 °C; and mycelium covers the plate after 8–9 days at 20 °C. A colony is densest, distinctly zonate, and grass green to spearmint green; mycelia are conspicuously dense; and surface hyphae form radial strands (Fig. 8c). Aerial hyphae are long and dense and form greenish aggregates that are granular to pulvinate (Fig. 8i). No diffusing pigment and odor. Chlamydospores examined after 7 days are 5.5–9 × 5–7.5 μm ((overline{x }) = 7 ± 0.9 × 6 ± 0.6, n = 30), globose to oval when terminal, and smooth, with an almost unobserved intercalary (Fig. 8k).On SNA, growth is approximately 7 mm/day at 20 °C and 5 mm/day at 25 °C; and mycelium covers the plate after 10 days at 20 °C. A colony is hyaline, thin, and loose, with little mycelium on the agar surface, not or indistinctly zonate, but becomes zonate by conidiation in white tufts after 4–5 days; and the margin is downy by long aerial hyphae, which degenerating/dissolving soon (Fig. 8d).Trichoderma pyramidale W. Jaklitsch & P. Chaverri, Mycologia 107: 581, 2015 (Fig. 9).Figure 9Cultures and asexual morph of Trichoderma pyramidale. (a–d) Cultures at 25 °C [(a) on CMD, 7 days; (b) on MEA, 4 days; (c) on PDA, 4 days; and (d) on SNA, 4 days]. (e) Conidiation tuft (PDA, 7 days). (f–j) Conidiophores and phialides (CMD, 5–7 days). (k) Conidia (CMD, 6 days). (l) Chlamydospores (PDA, 7 days). Scale bars: (e) = 2 mm; (f–l) = 10 μm.Full size imageDescription: Cultures and anamorph: optimal growth at 25 °C on all media, with little growth at 35 °C. On CMD, after 72 h, growth is 29–32 mm at 20 °C and 48–53 mm at 25 °C; and mycelium covers the plate after 5–6 days at 25 °C. Colonies show distinct zonation. Mycelium is loose and thin; hyphae are narrow, sinuous, and often form strands on the margin (Fig. 9a). Aerial hyphae are slight, forming a thin white to green downy, fluffy or floccose mat. Brown pigment is shown, but no distinct odor noted. Conidiophores are hyaline and thick with side branches on several levels at the base of the elongations that are mostly paired and at right angles with phialides in whorls of 3–5. Phialides are 5–9.5 × 2.5–3 μm ((overline{x }) = 7 ± 1.1 × 3 ± 0.3, n = 50) and 1–2.5 μm ((overline{x }) = 1.5 ± 0.3) wide at the base and often short, wide, and ampulliform (Fig. 9f–j). Conidia are 2.5–4 × 2.5–3.5 μm ((overline{x }) = 3.5 ± 0.3 × 3 ± 0.2, n = 50), green, smooth, and ellipsoidal (Fig. 9k).On PDA, after 72 h, growth is 41–43 mm at 20 °C and 50–55 mm at 25 °C; and mycelium covers the plate after 5–6 days at 25 °C. Colonies show indistinct zonation. Mycelia are dense, opaque, and thick; hyphae are wide, sinuous and often form strands on the margin (Fig. 9c). Margin is thin and ill defined. Aerial hyphae are slight and form a thin white to green downy, fluffy or floccose mat (Fig. 9e). Chlamydospores examined after 7 days are 5.5–10 × 5.5–10 μm ((overline{x }) = 7 ± 0.9 × 7 ± 0.9, n = 30), globose to oval when terminal or intercalary, and smooth (Fig. 9l).On SNA, after 72 h, growth is 33–35 mm at 20 °C and 38–40 mm at 25 °C; and mycelium covers the plate after 7–8 days at 25 °C. Colonies show distinct zonation. Mycelium is thin, yellow to green; hyphae are wide, sinuous, with indistinct strands on the margin (Fig. 9d). Margin is thin and ill defined. Aerial hyphae are slight and form a thin white downy, fluffy or floccose mat in distal parts of the colony. No diffusing pigment or distinct odor noted. Conidiation similar to CMD.Trichoderma rossicum Bissett et al., Canad. J. Bot. 81: 578, 2003 (Fig. 10).Figure 10Cultures and asexual morph of Trichoderma rossicum. (a–d) Cultures after 7 days at 25 °C [(a) on CMD; (b) on MEA; (c) on PDA; and (d) on SNA]. € Conidiation tuft (PDA, 7 days). (f–h,j,k) Conidiophores and phialides (CMD, 5–7 days). (i) Elongations (CMD, 6 days). (l,n) Conidia (CMD, 6 days). (m) Chlamydospores (PDA, 7 days). Scale bars: (e) = 2 mm; (f–n) = 10 μm.Full size imageDescription: Cultures and anamorph: optimal growth at 25 °C on all media. On CMD, growth of 10–11 mm/day at 20 °C and 15–17 mm/day at 25 °C; and mycelium covers the plate after 6–7 days at 20 °C. Colony is dense with a wavy margin, and the surface becomes distinctly zonate (Fig. 10a). Aerial hyphae are numerous, long, elongate, and villiform in the plate (Fig. 10i). No diffusing pigment or odor. Autolytic activity is variable, and coilings are scarce or inconspicuous. Conidiation noted after 3–4 days at 20 °C. Conidiation is effuse and in intense tufts that are hemispherical or irregular, and they show wide wheel grain banding that is gray green to deep green. Conidiophores radiate from the reticulum and are broad, straight, sinuous or helically twisted, show distally slightly pointed elongations, taper from the main axes to top branches, and present primary branches arranged in pairs or in whorls of 2–3, with secondary branches to solitary. Phialides are 4.5–14 × 2.5–4 μm ((overline{x }) = 7 ± 1.5 × 3.5 ± 0.3, n = 50) and 2–3.5 μm ((overline{x }) = 3 ± 0.4) wide at the base, ampulliform, and in whorls of 3–6 (Fig. 10f–h,j,k). Conidia are 3.5–5.5 × 2.5–4 μm ((overline{x }) = 4.5 ± 0.5 × 3 ± 0.2, n = 50), short cylindrical, and a gray color when single and pea green to yellow green in a group (Fig. 10l,n).On PDA, growth is 12–15 mm/day at 20 °C, 12–16 mm/day at 25 °C; and mycelium covers the plate after 4–5 days at 25 °C. Colony is denser with a wavy margin than that on CMD, and the surface is distinctly zonate (Fig. 10c). Aerial hyphae are numerous, long, and villiform to pulvinate in the plate. No diffusing pigment and odor (Fig. 10e). Autolytic activity is variable, coilings are scarce or inconspicuous. Chlamydospores examined after 7 days are 6.5–9.5 × 6–9 μm ((overline{x }) = 7 ± 1.0 × 7 ± 0.9, n = 30), terminal and intercalary, globose or ellipsoidal, and smooth (Fig. 10m).On SNA, growth is 8–13 mm/day at 20 °C and 8–12 mm/day at 25 °C; and mycelium covers the plate after 6–7 day at 25 °C. Colony is hyaline, thin and dense; and mycelium degenerate rapidly (Fig. 10d). Aerial hyphae are inconspicuous, autolytic activity is scant, and coilings are distinct. Conidiation noted after approximately 4 days and starts in white fluffy tufts spreading from the center to form concentric zones, and they compact to pustules with a white to greenish color. More

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Data sourcesThis study builds on two datasets; 666 livestock anthrax outbreaks collected over 60 years (1957–2017) by the Kenya Directorate of Veterinary Services (KDVS), and 13 reported anthrax outbreaks we investigated between 2017 and 201811,13. These datasets were combined with data from targeted active anthrax surveillance we conducted in 2019–2020 (see below) to define anthrax suitable areas in Kenya, including hotspots, and subsequently assessed effectiveness of livestock vaccination as a control strategy.Targeted active surveillance-collected anthrax data, 2019–2020Active anthrax surveillance was conducted for 12 months between 2019 and 2020 in randomly selected areas to ensure representation of all AEZs of the country. AEZs are land units defined based on the patterns of soil, landforms and climatic characteristics. Kenya has seven AEZs that include agro-alpine, high potential, medium potential, semi-arid, arid, very-arid and desert. In 2013, Kenya devolved governance into 47 semi-autonomous counties that are subdivided into 290 subcounties which are in turn divided into 1450 administrative wards, the smallest administrative units in the country. Using a geographic map that condensed Kenya into five AEZs; agro-alpine, high potential, medium potential, semi-arid, and arid/very arid zones, we randomly selected 4 administrative sub-counties from each AEZ (N = 20). To increase geographic spread of the study and enhance detection of anthrax outbreaks, we surveilled the larger administrative county (consisting of 20 to 45 administrative wards) where the randomly selected sub-counties were located. As shown in Fig. S1, we ultimately carried out the active anthrax surveillance in 18 counties, containing 523 administrative wards, the latter being used for measuring spatial association (see below).We conducted the surveillance between April 2019 and June 2020, through 523 animal health practitioners (AHPs), one in each ward, after intensive training to identify anthrax using a standard case definition, and to collect and electronically transmit the data weekly using telephone-based short messaging system (SMS) to a central server hosted by KDVS. Regarding case definition, any livestock death classified as anthrax through clinical or laboratory diagnosis was considered an anthrax event. Using standard guidelines issued by the KDVS, a clinical diagnosis was made by the AHPs across the country as an acute cattle, sheep or goat disease characterized by sudden death with or without bleeding from natural orifices, accompanied by absence of rigor mortis. Further, if the carcass was accidentally opened, failure of blood to clot and/or the presence of splenomegaly were included. In pigs, symptoms included swelling of the face and neck with oedema. A laboratory confirmed anthrax was diagnosed using Gram and methylene blue stains followed by identification of the capsule and typical rod-shaped B. anthracis in clinical specimens that the AHPs submitted to the central or regional veterinary investigation laboratories in Kenya. One case of anthrax in either species was considered an outbreak.During the surveillance, the programmed server sent prompting texts directly to the AHPs’ cell phones every Friday of each week for the 52 weeks. The AHPs interacted with the platform by responding to prompting questions sent via SMS to their telephones. Data were securely stored in an online encrypted platform which was subsequently downloaded into Ms Excel for analysis. This surveillance detected 119 anthrax outbreaks, whose partial data were used to model effects of climate change on future anthrax distribution in Kenya14. Here, we integrated these active surveillance data with other datasets to conduct detailed ENM and kernel-smoothed density mapping with a goal of refining suitable anthrax areas including crystalizing hotspots in the country.Anthrax outbreak incidence per livestock population by countyWe knew the total number of livestock per county and wards by species for the active surveillance period. The counties represented the level of disease management including vaccine distribution while the wards within counties represented the modeling unit for targeting control. Therefore, we estimated the outbreak incidence as the total number of outbreaks per livestock species per 100,000 head of that species.Ecological niche modeling and validationWe used boosted regression tree (BRT) algorithm as previously published13. In those studies, we estimated the geographic distribution of anthrax in southern Kenya using 69 spatially unique outbreak points (thinned from the 86 outbreaks in the records) and 18 environmental variables resampled to 250 m resolution. In this study, the final experiments were run with a learning rate (lr) = 0.001, bagging fraction (br) = 5, and maximum tree = 2500. We then mapped anthrax suitability as the mean output of the 100 experiments and the lower 2.5% and upper 97.5% mapped as confidence intervals. We determined variable contribution and derived partial dependence as previously described13. As BRTs are a random walk and each experiment randomly resamples training and test data, it was necessary to repeat those outputs along with the map predictions.Here, our goal was to evaluate the BRT models built with records data from 2011 to 2017 data and use the predict function to calculate model accuracy metrics using the 2017–2020 outbreaks as presence points and the sub-counties reporting zero outbreaks during the 2019–2020 active surveillance period as absence points. The model of southern Kenya was projected onto all of Kenya using climate variables clipped to the whole of Kenya. We tested the BRT models in two ways; first, evaluating 2011–2017 data models with holdout data using a random resampling and multi-modeling approach. Here, we report the area under curve (AUC) for each of the original training/testing split into the 69 historical points and the 2017–2020 data serving as independent data, the latter representing true model validation. Second, to determine the total percentage of surveillance data predicted and map areas of anthrax suitability to compare with kernel density estimates (see below), we produced a dichotomized map using the Youden index cutoff17 following Otieno et al.14.Outbreak concentrations from kernel density estimation (KDE)To describe the spatial concentration of reported outbreaks, we calculated descriptive spatial statistics, including the spatial mean, standard distance, and standard deviational ellipse of outbreak locations from the prospective surveillance dataset following Blackburn et al.18 These spatial statistics help to differentiate the geographic focus (spatial mean) and dispersion of outbreak reports from year to year and across the sampling period. We then conducted kernel density estimation (KDE) to visualize the concentration of anthrax outbreaks per square kilometer per year and across the study period18. We used the spatstat package for all KDE analyses using the quadratic kernel function19:$$fleft( x right) = frac{1}{{nh^{2} }} mathop sum limits_{i = 1}^{n} Kleft( {frac{{x – X_{i} }}{h}} right)$$where h is the bandwidth, x-Xi is the distance to each anthrax outbreak i. Finally, K is the quadratic kernel function, defined as:$$Kleft( x right) = frac{3}{4}left( {1 – x^{2} } right), left| x right| le 1$$$$Kleft( x right) = 0,x > 1$$This function was employed to estimate anthrax outbreak concentration across space using each outbreak weighted as one. We calculated the bandwidth (kernel) using hopt that uses the sample size (number of outbreaks) and the standard distance to estimate bandwidth. Finally, we estimated bandwidth for each year and then averaged them to apply the same fixed bandwidth for each year under study in Q-GIS version 3.1.8. The resulting outputs were map surfaces representing the spatial concentrations of outbreaks across the country per 1 km2 for each study year and all study years combined. For this study, we used the cutoff criteria of Nelson and Boots19 to identify outbreak hotspots as areas with density values in the upper 25%, 10%, and 5% of outbreak concentrations. The analyses identified these areas by year (2017–2020) and for all surveillance years combined.Local spatial clustering at the ward levelAnthrax outbreak incidence per livestock speciesThe ENM and KDE-derived maps provide a first estimate of potential risk and outbreak concentration, respectively. We were also interested in estimating anthrax outbreak intensity relative to livestock populations at a local level. For the active surveillance period, we knew the total number of outbreaks per ward (the smallest administrative spatial unit) by livestock species. For this two-year period, we estimated the ward-level outbreak incidence as the total number of outbreaks per livestock species per 10,000 head of that species. To estimate livestock population per ward, we extracted the values in the raster file of the areal weighted gridded livestock of the world data using the zonal statistic routine in Q-GIS version 3.1.8, into the polygon consisting of all pixels per ward as the total population19,20. We calculated outbreak incidence as the number of outbreaks per ward cattle population per 10,000 cattle for each administrative ward. We limited this analysis to those 18 counties participating in the active surveillance study (Fig. S1), as we could appropriately assume any ward with no reports was a ‘true zero’ for the estimation. Given that most reported outbreaks were in domestic cattle (see results below), we here report those results involving cattle alone. Given the overall high number of wards and the high number of wards without outbreaks, we performed the empirical Bayes smoothing and spatial Bayes smoothing routines in GeoDa version 1.12.1.161 to reduce the variance in anthrax incidence estimates20,21. To evaluate smoothing routine performance, we box plotted rates per ward and selected the method with the greatest reduction in outliers21. Smoothed rates were mapped as choropleth map in Q-GIS version 3.1.8 using the four equal area bins.Spatial cluster analysisWe used Local Moran’s I16 to test for spatial cluster of livestock anthrax in cattle using the smoothed outbreak incidence estimates. The Local Moran’s I statistic tests whether individual wards are part of spatial cluster, like incidence estimates surrounded by similar estimate (high-high or low-low) or spatial outliers where wards with significantly high or low estimates are surrounded by dissimilar values (high-low or low–high). The local Moran’s I is written as16:$$I_{i} = Z_{i} sum W_{ij} Z_{j}$$where Ii is the statistic for a ward i, Zi is the difference between the incidence at i and the mean anthrax incidence rate for all of wards in the study, Zj is the difference between anthrax risk at ward j and the mean for all wards. Wij is the weights matrix. In this study, the 1st order queen contiguity was employed. Here, Wij equals 1/n if a ward shared a boundary or vertex and 0 if not. For this study, Local Moran’s I was performed on the wards using 999 permutations and p = 0.05 using GeoDa version 1.12.1.161.Assessing effectiveness of cattle vaccination in burden hotspotsAs a first estimate of how we might scale up livestock anthrax vaccination efforts in Kenya, we slightly adjusted a simple published anthrax outbreak simulation model in a cattle population. For this study we applied an early mathematical approach of Funiss and Hahn22 to simulate anthrax at the ward level. While other recent models are available23,24, these are difficult to parameterize or require time series data we could not derive with the surveillance approach in this study. Like the more recent models, Funiss and Hahn22 assumed anthrax transmission was driven by cattle accessing spore-contaminated environments. Here the proportion of infected cattle each day depended on the population of susceptible animals in the population and probability of getting infected. This probability depends on environmental contamination (“a”), and a fraction of anthrax carcasses in the environment on a day (“f,”). Each day, the newly infected cattle are transferred to an incubation period vector, “d,” waiting to die following a probability “p”. In this model, all infected animals, “n,” die following the incubation periods given by the vector, “p”, in which pi is the probability of a cow dying i days after the infection. Following death, the cattle are transferred to a carcass state, providing a direct infection source to the susceptible cattle via environmental contamination. Environmental contamination “a,” is therefore defined as the number of spores ingested by an animal in a day. This environmental contamination depends on spores from carcasses and an assumed spore decay rate γ22.The complete set of difference equations with a daily time step is given by:$${text{S}}_{(t + 1)} = {text{S}}_{(t)} – {text{ S}}_{(t)} *left( {{1} – {text{e}}^{{ – left( {{text{a}}_{t} + gamma {text{f}}_{{{text{t}} + 1}} } right)}} } right)$$$${text{I}}_{(t + 1)} = {text{I}}_{(t)} + {text{ S}}_{(t)} *left( {{1} – {text{e}}^{{ – left( {{text{a}}_{{text{t}}} + gamma {text{f}}_{{{text{t}} + {1}}} } right)}} } right)$$where the expression (left( {{1} – {text{e}}^{{ – left( {{text{a}}_{t} + gamma {text{f}}_{{{text{t}} + 1}} } right)}} } right)) denotes the probability of an animal becoming infected and at + γft+1 is the mean number of spores ingested by a cow in a day. The equation for environmental contamination, a, is given by:$${text{a}}_{t + 1} {-}{text{a}}_{{text{t}}} = alpha {text{a}}_{{text{t}}} + beta {text{c}}_{{{text{t}} + {1}}}$$The newly infected animals die after a certain number of days. The distribution of incubation periods is given by the vector, p. On each day, the new cases are placed in a due-to-die vector, d, and when they die, they are subsequently moved down one step to fresh carcasses, ft. The fresh carcasses provide a direct source of infection to the susceptible cattle via the ‘fresh carcass term’, γ. These carcasses decay or are scavenged or disposed by man. The equation expressing the disseminating carcasses, c, is:$${text{C}}_{t + 1} – {text{c}}_{t} = {text{f}}_{t + 1} – delta {text{c}}_{t}$$The model parameters variables are provided in Table 1 and are similar to those used by Funiss and Hahn22 to generate a standard run. We ran the model for one year and extrapolated to cattle population in the identified hotspot wards.Table 1 Model parameters and variables.Full size table More