Truffle fungi enriched in cattle-grazed native pecan groves under NPK fertilization

Pecan (Carya illinoinensis [Wangenh.]K. Koch) a major tree nut crop native to North America, with 34Oklahoma contributing nearly 80% U.S. native and seedling pecan groves typically lack structured orchard practices such as fertilization, irrigation, or herbicide 36 application.Instead, they depend on natural nutrient cycling.Among rhizosphere microbes, 37 ectomycorrhizal (ECM) fungi, including truffle-forming Tuber species-a group known for their ecological 38 and commercial value, form mutualistic associations with pecan roots and, in some species, produce edible 39 truffles [1, 2].Fertilization is known to restructure microbial communities, sometimes enhancing certain 40 ECM taxa while suppressing others [3].However, the response of ECM fungi, particularly Tuber, to 41 fertilization remains largely unexplored in native pecan groves.Here we report evidence that cattle grazing 42 combined with NPK fertilization promotes the enrichment of Tuber species, including the pecan truffle 43 (Tuber lyonii), in Oklahoma native pecan groves.We analyzed fungal communities from roots and 44 rhizosphere soils of two native pecan groves with contrasting management: Grove A, incorporated cattle 45 grazing while Grove B incorporated hay production but not grazing.In each grove, two fertilizer treatments 46 (Full NPK consisting of 300lbs/acre fertilizer in ratio of 11N:27P:27K versus No NPK fertilizer) were 47 utilized, and roots and rhizosphere soils were sampled prior to fertilization in February and after fertilization 48 in May and September 2023.Total DNA was extracted and sequenced (Illumina MiSeq).The sequences 49were processed with Mothur (v.1.48)[4] and R (v4.4.1).Relative abundances of fungal taxa were quantified 50 at genus and species levels, with emphasis on ectomycorrhiza, particularly the genus Tuber.Fungal 51 communities showed strong variation across groves, fertilization, and season.In Grove A (cattle grazing), 52Tuber and Russula dominated roots, while Grove B (hay) roots were dominated by Inocybe and Russula. 53In Grove A roots in September under Full NPK, relative abundance of Tuber (~20%) was higher compared 54 to No NPK (~9%) (Fig. 1A).Although Tuber abundance was lower in roots from Grove B, NPK fertilization 55 increased it's abundance in the rhizosphere soil (Fig. 1B).Fertilization boosted Tuber abundance in both 56 groves, but the effect was most pronounced in grazed systems, suggesting that grove legacy interacts with 57 nutrient inputs to shape ECM composition (Fig. 1C).Species-level resolution revealed five Tuber taxa: T. 58 lyonii, T. walkeri, T. floridanum, T. brennemanii, and T. mexiusanum (Fig. 1A).Among these, T. walkeri was 59 the most widespread across groves, while T. lyonii was concentrated in Grove A roots and increased under 60 Full NPK during September, aligning with its typical fruiting season.T. floridanum was also favored in 61 Grove A, while T. mexiusanum was enriched in Grove B under No NPK, indicating their sensitivity to 62 fertilization.T. brennemanii appeared more tolerant of nutrient additions, persisting with or without NPK 63 fertilization.These species-specific responses demonstrate ecological filtering within native pecan systems. 64Functional guild analysis confirmed ECM fungi as the dominant group in both native pecan roots and soils 65 (Fig. 1C), consistent with the well-established role of ECM associations with improved pecan roots [4].In 66 roots, ECM abundance was only slightly higher under Full NPK (41%) than No NPK (37%) (non-67 significant).Soil showed significant variation, overall, with higher ECM in Grove B and Full NPK 68 treatment.Seasonal variation was also evident: in Grove A, T. lyonii and T. walkeri peaked in September, 69coinciding with late-season carbon allocation.This suggests that fertilizer inputs, grazing, and seasonal host 70 physiology jointly influenced truffle colonization dynamics.valued for cattle and nut production, to be managed for cattle, nuts and truffles.Such co-production 75 diversify growers' income and align native pecan systems with European truffle-orchard models.76 Management species-specific: T. lyonii and T. walkeri mexiusanum 77 in unfertilized refugia.Tailoring fertilization could optimize both truffle biodiversity 78 and pecan yield.Our findings suggest that under the right grove management fertilization can enhance 79 desirable truffle taxa, consistent with recent reports that moderate nitrogen inputs may favor symbiotrophic 80 fungi [3].This expands our understanding of nutrient-fungi-host interactions and demonstrates how 81 management legacies condition microbial responses.Fostering ECM fungi, particularly truffles, may 82 enhance both ecological functions, such as nutrient acquisition, stress resilience, and economic outcomes 83 through potential co-harvest of nuts and truffles.In conclusion, we provide the first report of species-84 specific Tuber enrichment in pecan groves, indicating that cattle grazing and NPK fertilization may 85 foster conditions favorable for pecan truffle production.Grove A, the grazed system, supported higher T. 86 lyonii and T. walkeri abundance, particularly in fertilized roots during September.These results highlight 87 the potential for integrating truffle co-cultivation into native pecan management, representing a novel 88 horticultural innovation.Future research should confirm truffle fruiting in these groves, evaluate 89 inoculation strategies, and quantify economic returns from dual-use pecan-truffle systems.