Climate-Smart \u3cem\u3eBrachiaria\u3c/em\u3e for Improving Livestock Production in East Africa: Emerging Opportunities

Brachiaria grass is an important tropical forage of African origin with desirable attributes of agricultural and environmental significance. Brachiaria has been extensively cultivated as a pasture across the tropics except in its endemic provenance of Africa. In 2013, a collaborative research program was initiated in Kenya and Rwanda with the aim of improving the availability of quality livestock feeds adapted to drought and low fertility soils using Brachiaria. The outcomes sought were increased livestock productivity leading to improved farmer income and the development of seed production opportunities. The program has identified five preferred cultivars, and four of them are currently being evaluated on-farm by over 2000 small-holder farmers in Kenya and Rwanda for livestock productivity. Preliminary milk production data has shown a 15 to 40% increase in milk production in Kenya and an average increase of 36% in Rwanda. The substitution of Napier grass by Brachiaria in the feed has increased average daily body weight gain of cattle by 205g during a 12 week period. Kenyan farmers reported increased on-farm forage availability by three months after Brachiaria introduction. The program has also worked to determine the role of endophytes and plant associated microbes for the improvement of biomass production and adaptation of Brachiaria to biotic and abiotic stresses. A diverse group of fungi and bacteria were isolated, identified and characterized, and the role of these microbes on plant growth and plant pathogen suppression is being investigated. This paper discusses the rationale for selecting Brachiaria as potential forage for eastern Africa and highlights current achievements, and identifies areas for future research

Investigations of ectomycorrhizal fungal communities in terrestrial and canopy environments

Methodologies to detect ectomycorrhizal fungi from below-ground environments, including canopy soil environments, and canopy root formation were investigated in native and non-native vegetation of New Zealand. The methodologies tested include both molecular (DNA sequence barcoding and fingerprinting analyses) and non-molecular (hyphal ingrowth bags, ergosterol assays and artificial environment manipulations) approaches. Soil diversity of Cortinarius (an ectomycorrhizal fungal genus) communities associated with native (Nothofagus spp. and Kunzea ericoides) and exotic (Pinus radiata) ectomycorrhizal hosts were compared using genus-specific sequence barcoding. Analysis of the soil-clones detected from the three ectomycorrhizal hosts identified all clones were from the genus Cortinarius and covered a range of the genus’ large diversity. A statistical comparative measure of the 138 soil-clones showed the Kunzea and Pinus soil-clone communities were significantly different from the Nothofagus community but were not significantly different from each other. Thirty-one unique molecular operational taxonomic units (MOTUs) were described from the soil-clone sequences, 3 MOTUs were shared between all hosts; Pinus and Kunzea shared 9 MOTUs, while only sharing 1 MOTU each with Nothofagus. The soil-clone MOTU richness was compared with taxonomic richness of Cortinarius species for the three hosts in New Zealand. The 11 MOTUs found from N. menziesii and N. solandri var. cliffortioides is, as expected, much lower than the 176 Cortinarius species recorded in association with all five Nothofagus species throughout New Zealand. In contrast, the 21 MOTUs collected from K. ericoides are greater than the 14 Cortinarius species currently recorded in association with this host nationwide. Sixteen unique MOTUs were described from the P. radiata soil-clones, while there are no published records of Cortinarius in association with P. radiata in New Zealand. The DNA fingerprinting technique terminal restriction fragment length polymorphism (TRFLP) analysis was investigated to determine accuracy of detecting ectomycorrhizal fungal species richness from pools of mixed-species DNA. Mixed-species samples (pools) with low known species richness (total species ≤ 10) were estimated at ~ 2–12 times greater by TRFLP analysis, and pools with high species richness (total species ≥ 19) were estimated at 0.6–1 times lower by the analysis. The raw data show that the high variation observed from replicates of each enzyme is predominantly caused by disagreement between the TRFs detected by the 6FAM and PET dyes; the two dyes represent the terminal ends of sequence fragments amplified by the primers ITS1F (6FAM) and ITS4 (PET). Intragenomic variation within the ITS region of ectomycorrhizal sporocarps is the probable cause for the observed high variation in TRF profiles, especially in causing overestimation of low richness. Accumulation into hyphal ingrowth bags of hyphal diversity and mass of ectomycorrhizal fungi associated with Nothofagus spp. was investigated over a period of 10 months, with a combined approach of molecular (TRFLP analysis) and non-molecular (ergosterol assay) analysis. Fungal mass (measured as ergosterol per gram of sand) extracted from the incubated hyphal ingrowth bags was found to significantly increase over a period of 10 months (from 0.1–5.2 μg g-1). Ectomycorrhizal fungal diversity and richness also increased over time (from 106– 187 unique TRFs), although this relationship was not significant. Ordination and cluster analyses show that there are no time dependent groupings of the ectomycorrhizal fungal communities, showing that while diversity and richness increase over time, the communities of TRFs (i.e., effective species) were not greatly altered. Thus, species succession was not observed from the accumulation of ectomycorrhizal fungi in hyphal ingrowth bags. The accumulation of humus soil in a canopy environment (along the upper surface of branches) was investigated as the cue for canopy root growth in native New Zealand N. menziesii and N. solandri var. cliffortioides. After an application of “artificial canopy soil” over a period of 12 months, no canopy root growth was observed in either of the Nothofagus host trees. This shows that (i) accumulation of soil alone is not the cue for canopy root growth, (ii) the application period (max 12 months) was not long enough to allow for root growth, or (iii) multiple factors cue the growth of canopy roots in Nothofagus systems. Below-ground ectomycorrhizal fungal communities associated with canopy and terrestrial root systems of N. menziesii were investigated using TRFLP analysis and ergosterol assays on hyphae accumulated in hyphal ingrowth bags. The diversity and richness of ectomycorrhizal fungi was greater in the terrestrial root systems in comparison to the canopy environments. The TRF communities from the terrestrial environment were highly diverse, while the canopy communities were very similar to each other. However, the canopy and terrestrial TRF communities were shown to share some ectomycorrhizal fungi (27% of TRFs), showing that while the terrestrial communities are more species rich and diverse there are some ectomycorrhizal fungi that are present in both communities. This thesis concluded that the TRFLP analysis method proposed was more appropriate than the sequence barcoding protocol with genus-specific primers. While both methods had limitations, TRFLP analysis descriptions of below-ground ectomycorrhizal fungal communities were of greater benefit. Hyphal ingrowth bags were shown to accumulate ectomycorrhizal fungal communities without an effect of incubation time on the detected communities. The previously proposed cue initiating canopy roots growth was concluded to not be the only influencing factor in Nothofagus species. Finally, Nothofagus canopy ectomycorrhizal fungal communities were similar independent of the tree host and were less diverse than terrestrial communities, although all communities were found to share some fungi

Investigations of ectomycorrhizal fungal communities in terrestrial and canopy environments

Methodologies to detect ectomycorrhizal fungi from below-ground environments, including canopy soil environments, and canopy root formation were investigated in native and non-native vegetation of New Zealand. The methodologies tested include both molecular (DNA sequence barcoding and fingerprinting analyses) and non-molecular (hyphal ingrowth bags, ergosterol assays and artificial environment manipulations) approaches. Soil diversity of Cortinarius (an ectomycorrhizal fungal genus) communities associated with native (Nothofagus spp. and Kunzea ericoides) and exotic (Pinus radiata) ectomycorrhizal hosts were compared using genus-specific sequence barcoding. Analysis of the soil-clones detected from the three ectomycorrhizal hosts identified all clones were from the genus Cortinarius and covered a range of the genus’ large diversity. A statistical comparative measure of the 138 soil-clones showed the Kunzea and Pinus soil-clone communities were significantly different from the Nothofagus community but were not significantly different from each other. Thirty-one unique molecular operational taxonomic units (MOTUs) were described from the soil-clone sequences, 3 MOTUs were shared between all hosts; Pinus and Kunzea shared 9 MOTUs, while only sharing 1 MOTU each with Nothofagus. The soil-clone MOTU richness was compared with taxonomic richness of Cortinarius species for the three hosts in New Zealand. The 11 MOTUs found from N. menziesii and N. solandri var. cliffortioides is, as expected, much lower than the 176 Cortinarius species recorded in association with all five Nothofagus species throughout New Zealand. In contrast, the 21 MOTUs collected from K. ericoides are greater than the 14 Cortinarius species currently recorded in association with this host nationwide. Sixteen unique MOTUs were described from the P. radiata soil-clones, while there are no published records of Cortinarius in association with P. radiata in New Zealand. The DNA fingerprinting technique terminal restriction fragment length polymorphism (TRFLP) analysis was investigated to determine accuracy of detecting ectomycorrhizal fungal species richness from pools of mixed-species DNA. Mixed-species samples (pools) with low known species richness (total species ≤ 10) were estimated at ~ 2–12 times greater by TRFLP analysis, and pools with high species richness (total species ≥ 19) were estimated at 0.6–1 times lower by the analysis. The raw data show that the high variation observed from replicates of each enzyme is predominantly caused by disagreement between the TRFs detected by the 6FAM and PET dyes; the two dyes represent the terminal ends of sequence fragments amplified by the primers ITS1F (6FAM) and ITS4 (PET). Intragenomic variation within the ITS region of ectomycorrhizal sporocarps is the probable cause for the observed high variation in TRF profiles, especially in causing overestimation of low richness. Accumulation into hyphal ingrowth bags of hyphal diversity and mass of ectomycorrhizal fungi associated with Nothofagus spp. was investigated over a period of 10 months, with a combined approach of molecular (TRFLP analysis) and non-molecular (ergosterol assay) analysis. Fungal mass (measured as ergosterol per gram of sand) extracted from the incubated hyphal ingrowth bags was found to significantly increase over a period of 10 months (from 0.1–5.2 μg g-1). Ectomycorrhizal fungal diversity and richness also increased over time (from 106– 187 unique TRFs), although this relationship was not significant. Ordination and cluster analyses show that there are no time dependent groupings of the ectomycorrhizal fungal communities, showing that while diversity and richness increase over time, the communities of TRFs (i.e., effective species) were not greatly altered. Thus, species succession was not observed from the accumulation of ectomycorrhizal fungi in hyphal ingrowth bags. The accumulation of humus soil in a canopy environment (along the upper surface of branches) was investigated as the cue for canopy root growth in native New Zealand N. menziesii and N. solandri var. cliffortioides. After an application of “artificial canopy soil” over a period of 12 months, no canopy root growth was observed in either of the Nothofagus host trees. This shows that (i) accumulation of soil alone is not the cue for canopy root growth, (ii) the application period (max 12 months) was not long enough to allow for root growth, or (iii) multiple factors cue the growth of canopy roots in Nothofagus systems. Below-ground ectomycorrhizal fungal communities associated with canopy and terrestrial root systems of N. menziesii were investigated using TRFLP analysis and ergosterol assays on hyphae accumulated in hyphal ingrowth bags. The diversity and richness of ectomycorrhizal fungi was greater in the terrestrial root systems in comparison to the canopy environments. The TRF communities from the terrestrial environment were highly diverse, while the canopy communities were very similar to each other. However, the canopy and terrestrial TRF communities were shown to share some ectomycorrhizal fungi (27% of TRFs), showing that while the terrestrial communities are more species rich and diverse there are some ectomycorrhizal fungi that are present in both communities. This thesis concluded that the TRFLP analysis method proposed was more appropriate than the sequence barcoding protocol with genus-specific primers. While both methods had limitations, TRFLP analysis descriptions of below-ground ectomycorrhizal fungal communities were of greater benefit. Hyphal ingrowth bags were shown to accumulate ectomycorrhizal fungal communities without an effect of incubation time on the detected communities. The previously proposed cue initiating canopy roots growth was concluded to not be the only influencing factor in Nothofagus species. Finally, Nothofagus canopy ectomycorrhizal fungal communities were similar independent of the tree host and were less diverse than terrestrial communities, although all communities were found to share some fungi

Detecting and identifying ectomycorrhizal fungi in New Zealand silver beech (Lophozonia menziesii, Nothofagaceae) forest: a case study

Adventitious roots in canopy soils associated with silver beech (Lophozonia menziesii (Hook.f.) Heenan & Smissen (Nothofagaceae)) form ectomycorrhizal associations. We used amplicon sequencing of the internal transcribed spacer 2 region to compare diversity of ectomycorrhizal fungal species in canopy and terrestrial sites. The study data are archived as an NCBI BioProject (accession PRJNA421209), with the raw DNA sequence reads available from the NCBI Sequence Read Archive SRA637723 Community composition of canopy ectomycorrhizal fungi was significantly different to the terrestrial community composition, with several abundant ectomycorrhizal species significantly more represented in the terrestrial soil than the canopy soil. Additionally, we found evidence that an introduced ectomycorrhizal species was present in these native forest soils. We identified OTUs in two ways: (i) by manually curated BLAST searching of the NCBI nr database, and (ii) by comparison with Species Hypotheses on UNITE v.7.2. We desired to make species identifications where we could be reasonably confident they were robust, but had to avoid making identifications when an incorrect name could have implications for biosecurity or our understanding of biodiversity and biogeography. We found some UNITE Species Hypotheses included sequences of more than one taxon, which we were able to separate and distinguish by phylogenetic analysis. Consequently we exercised caution in reporting names based on the Species Hypotheses. Using data from this case study, we will illustrate the achievements and challenges faced in identifying species of ectomycorrhizal fungi from DNA barcodes. Most DNA sequences of ectomycorrhizal fungi matched closely New Zealand voucher specimens stored in either the New Zealand Fungal Herbarium (PDD) or the Otago Regional Herbarium (OTA), which facilitated the validation of identifications. In the case of PDD specimens, collection and DNA data were linked via the Systematics Collections Data database (https://scd.landcareresearch.co.nz). We are working towards a similar database for OTA specimens, using the Specify 6 database platform

Fungal diversity in canopy soil of silver beech, Nothofagus menziesii (Nothofagaceae).

Adventitious roots in canopy soils associated with silver beech (Nothofagus menziesii Hook.f. (Nothofagaceae)) form ectomycorrhizal associations. We investigated the extent to which canopy ectomycorrhizal communities contribute to overall diversity of ectomycorrhizal fungi associated with silver beech. Hyphal ingrowth bags were buried for 12 months in canopy and terrestrial soils of five trees at one site. We used amplicon sequencing of the nuclear ribosomal internal transcribed spacer 2 region (ITS2) to assess diversity of both ectomycorrhizal and non-ectomycorrhizal OTUs in hyphal ingrowth bags. There was a significant difference in ectomycorrhizal fungal community diversity between the terrestrial and canopy hyphal ingrowth bag communities. Ectomycorrhizal community composition of the terrestrial and canopy environments was also significantly different. Some ectomycorrhizal taxa were significantly differentially represented in either the terrestrial or canopy environment. The hyphal ingrowth bags also accumulated non-ectomycorrhizal species. The non-ectomycorrhizal fungi also had significantly different diversity and community composition between the canopy and terrestrial environments. Like the ectomycorrhizal community, some non-ectomycorrhizal taxa were significantly differentially represented in either the terrestrial or canopy environment. The canopy soil microhabitat provides a novel environment for growth of ectomycorrhizal adventitious roots and enables the spatial partitioning of ectomycorrhizal and non-ectomycorrhizal fungal diversity in the forest