Determining How the Ubiquitous Fungi Mortierella Regulates Belowground N Dynamics Under Different Crop Rotation Systems

1. Identifying the ideal substrate and condition for cotton growth

To mitigate the effects of external microbes on cotton performance, we performed surface sterilization of cotton seeds (Deltapine® 1646B2XF and PHY 400 W3FE) before germinating. Specifically, seeds were soaked in distilled de-ionized (DDI) water overnight and then soaked in 10% bleach for 20 min and rinsed with DDI water twice. Afterward, they were soaked in 2% H₂O₂ for 30 min and rinsed with sterilized DDI water twice. Surface-sterilized cotton seeds were transferred into plastic pots containing autoclaved sand in a growth chamber with 25 °C and 70% humidity and exposed to fluorescent LED lights (200 μmole m⁻² s⁻¹) for 12 h per day (Fig. 1A and B). After 14 days of germination (1^st true leaf stage), cotton seedlings were transplanted into individual pots containing sterilized sand under the same condition (Fig. 1C).

2. Isolating chitin consumers from sod-based rotation (SBR) soils using crab shells

To isolate the native nitrogen (N)-associated fungi as the inocula in this study, we used crab/lobster shell as the chitin substrate (one of the most important organic N sources in the agroecosystem) to culture chitin-consuming fungi (e.g., Mortierella spp.) from sod-based rotation (SBR; bahiagrass-bahiagrass-cotton-peanut) field soil. Specifically, we collected soil from cotton plots under the SBR system. The soil samples were then sieved through a screen with 0.64 cm apertures to achieve a more uniform soil particle size. We sprinkled and embedded flakes of sterilized crab shells in the fresh soil samples and incubated them in petri dishes. After 3, 5, and 7 days of incubation, we pulled some shells and plated very well-washed shells on solid Modified-Melin-Norkrans (MMN) media. Fig. 2 shows the isolated fungal strains.  

3. Seting up an in-vitro study (culture-based) and applying the RNAseq approach to identify the key genes of representative chitin consumers (e.g., Mortierella sp.) in response to the chitin substrate

To investigate molecular responses of M.elongata (PMI_93) to chitin, we cultured Mortierella in the MMN media with and without the chitin substrate. After 32 hours of culture, we extracted the RNA of PMI_93 fungal mycelia using a CTAB/chloroform extraction, and then the whole cDNA of each sample was sequenced using HiSeq 2000 (Illumina) in the Institute for Genome Sciences and Policy at Duke University. We obtained a total of 157,529,434 (26,254,906 on average) raw reads, and roughly 80% of reads were mapped to the M. elongata genome (M. elongata AG77v2; https://genome.jgi.doe.gov/portal/Morel2/Morel2.download.html). Venn diagram showed that the majority of genes (12,020 genes) were overlapped between Mortierella + chitin and Mortierella – chitin cultures, with only 317 and 312 genes being unique, respectively (Fig. 3A). Further, by performing differential gene expression analysis (cutoff values of |log2FoldChange| >1 and P value < 0.05), we found 239 differentially expressed genes (DEGs) following chitin substrate, with more up-regulated genes (177 genes) compared to down-regulated genes (62 genes; Fig. 3B). This implies that the chitin substrate resulted in a small difference in gene expression. However, principal component analysis of Mortierella genes showed that the expression pattern of these genes treated with the chitin substrate was clearly separated from that of genes without the chitin substrate (Fig. 3C), suggesting that the chitin substrate induced profoundly different expression patterns of whole Mortierella genes. 

By annotating DEGs to the EuKaryotic Orthologous Groups (KOG), we found that most up-regulated genes belonged to cellular processes and signaling (31%) and metabolism (31%; Fig. 4A), while over half of down-regulated genes were encoded to metabolism (56%; Fig. 4B). Within these two KOG groups, cell motility, cell wall/membrane/envelope biogenesis, and posttranslational modification, protein turnover, and chaperones were highly expressed in the chitin treatment. As expected, the chitin substrate promoted the expression of genes associated with chitin-related functions, including chitinase, chitin synthase/hyaluronan synthase (glycosyltransferases), and chitinase carbohydrate (Fig. 4C). These suggest that the chitin substrate may promote the growth and activity of Mortierella by providing an important component of cell wall–supporting polymers (e.g., polysaccharides) for the development of fungal cell walls and serving as the C and N source per se. Besides, some genes encoded to chromatin structure and dynamics, RNA processing and modification, transcription, as well as translation, ribosomal structure and biogenesis, were also highly expressed in the chitin treatment, suggesting that the chitin substrate improved the functioning of Moetierella genes involved in genetic information storage and processing. These functions ultimately can promote the development and turnover of Mortierella. 

4 Identifying the role of Mortierella elongata in plant performance via a greenhouse bioassay

Mortierella spp. have been demonstrated to have a dual endophyte–saprotroph lifestyle, which may exert plant-growth promotion to many crops via different mechanisms (Zhang et al., 2020). For example, Mortierella may interact with plants and function as synergistic agents, producing antibiotics and phytohormones (e.g., apocarotenoid, indoleacetic acid, and gibberellic acid) (Ozimek et al., 2018; Liao et al., 2019). Mortierella may serve as a saprotroph that drives soil carbon cycling (Clemmensen et al., 2013), P dissolution and immobilization (Detheridge et al., 2016), lipid metabolism, and chitin degradation (Uehling et al., 2017). The Fusarium oxysporum species complex, another major fungal species, comprises a variety of strains ubiquitously living in soils, including plant pathogenic and nonpathogenic strains (Edel-Hermann and Lecomte, 2019). Pathogenic F. oxysporum strains display high functional and genetic diversity (Steinberg et al., 2016), as evidenced by their wide range of plant hosts, including both dicots (e.g., bean, carnation, cotton, and tomato) and monocots (e.g., banana, orchids, and palms) (Edel-Hermann and Lecomte, 2019). However, the pathogenicity of F. oxysporum is host specific. Typically, pathogenic F. oxysporum strains develop two types of symptoms, i.e., vascular wilting that causes Fusarium wilt (a major disease) and root rot (Olivain and Alabouvette, 1999). Fusarium and Mortierella spp. are considered the dominant fungal species in agricultural soils and contribute over 0.5% of the relative abundance in total soil fungal communities. However, how plant-growth-promoting fungi (M. elongata) interact with putative pathogenic fungi (F. oxysporum) and how their interaction affects cotton growth are largely understudied. 

To address this question, we performed a greenhouse bioassay by inoculating M. elongate and F. oxysporum in two cotton cultivars (Deltapine® 1646B2XF and PHY400 W3FE). Two months after inoculation, we harvested cotton plants. Harvested plant samples were washed with DDI water twice and wiped by Kimtech Kimwipes Delicate Task Wipers (Kimberly-Clark Corporation, TX, USA). Fresh plant weight and plant height were measured immediately. To avoid biases caused by the different weights and heights among cotton seedlings, we used the changes in plant weight and heights before inoculation with fungi and after harvesting (weight/height_harvest- weight/height_inoculation) to represent plant performance. 

There was a significant interaction between fungal treatments and cultivars for changes in cotton biomass (Fig. 5A; P < 0.01). Fungal species only had an impact in the DP1646B2XF cultivar, where F. oxysporum and mixed inocula of M. elongata and F.oxysporum had the lowest cotton biomass change but M. elongata induced the highest cotton biomass change. In contrast, there was no significant difference among fungal treatments in the PHY400 W3FE cultivar, although fungal-inoculated treatments induced a slightly higher weight change as compared to the non-fungal treatment. Similarly, there was a significant interaction of fungal treatment by cultivars for cotton height change (Fig. 5B; P = 0.03). Fungal treatments only affected the height of the DP1646B2XF cultivar, where M. elongata led to the largest height change and mixed inocula had the lowest height change. A cultivar effect was observed in the mixed inocula inoculated treatment, with a significantly larger height change in PHY400 W3FE than DP1646B2XF. Overall, these results suggest M. elongata promoted cotton growth, consistent with our previous study showing that M. elongata exerted plant-growth promotion to many crops (Zhang et al., 2020). We also found that F. oxysporum may function as a pathogen in the DP1646B2XF but not in the PHY400 W3FE, demonstrating that the pathogenicity of F. oxysporum is host specific. The negative effect of the interaction of M. elongate and F. oxysporum on the performance of the DP1646B2XF cultivar suggests that the promoting effect of M. elongata was less than the detrimental effect of F. oxysporum on cotton growth. 

5. 5. Uncovering molecular mechanisms responsible for the role of chitin in the Mortierella-plant interaction using Metatranscripotimic analysis

To further unravel the effect of chitin addition on the Mortierella-plant interaction, we performed a Mortierella-plant-chitin greenhouse bioassay. Specifically, we inoculated M. elongata in popular seedling roots and then transplanted them into plastic pots that were filled with 180g of sterile play sand. For the chitin treatment, autoclaved crab shells were mixed with sand at a ratio of 2% (w/w). Plants were grown in a growth chamber at 25 °C and 70% humidity and exposed to fluorescent LED lights (200 μmole m⁻² s⁻¹) for 12 h per day (Fig. 1A and B). Four months after inoculation, we harvested plant root samples and extracted their total RNA using the CTAB/LiCl extraction method. The whole cDNA of root samples was sequenced using HiSeq 2000 (Illumina) in the Institute for Genome Sciences and Policy at Duke University. We obtained a total of 238,895,950 reads (34,127,993 on average) after filtering low-quality reads, and around 1% of reads were mapped to the M. elongata genome ( M. elongata AG77v2; https://genome.jgi.doe.gov/portal/Morel2/Morel2.download.html). A Venn diagram showed that chitin addition yielded fewer Mortierella genes (8,776 genes) than the no-chitin addition (10,343 genes), with most of the genes (8,166 genes) overlapping (Fig. 6A). Our differential gene expression analysis (with cutoff values of |log2FoldChange| >1 and P value < 0.05) revealed that chitin addition induced more up-regulated Mortierella genes (513 genes) than down-regulated genes (275 genes). This suggests that chitin addition can promote the expression of some genes despite fewer  Mortierella genes being detected. A PCA plot showed that there was a clear separation for the expression pattern of all Mortierella genes with and without chitin addition, suggesting that chitin addition can substantially induce Mortierella gene expression in plant roots. 

We further filtered highly DEGs with cutoff values of |log2FoldChange| > 1 and P value < 0.01 and annotated these genes using KOG (Fig. 7). Most up-regulated genes were mapped into the group of cellular processes and signaling, metabolism, and information storage and processing (Fig. 7A), while the majority of down-regulated genes were assigned to cellular processes and signaling as well as metabolism (Fig. 7B). Notably, chitin addition induced the high expression of genes associated with cytoskeleton, extracellular structures, nuclear structure, and posttranslational modification, protein turnover, chaperones. This implies that chitin addition promoted the cell development of Mortierella in the plant root. Besides, some highly expressed genes following chitin addition functioned as chromatin structure and dynamics, translation, ribosomal structure and biogenesis, and cell cycle control, cell division, chromosome partitioning, suggesting chitin addition can accelerate cell division and turnover and subsequently facilitate Mortierella growth in plant roots. We also found some highly expressed genes induced by chitin addition were encoded to carbohydrate transport and metabolism (especially three genes associated with chitinase; Fig. 7C), energy production and conversion, and inorganic ion transport and metabolism (including Ca²⁺ transporting ATPase, Halotolerance protein HAL3 (contains flavoprotein domain), and Na⁺/K⁺ ATPase, alpha subunit). N is essential for microorganisms and plants because it is a key constituent of biomolecules including DNA, RNA, chlorophyll, and enzymes (Daly et al., 2021). Our results demonstrate that chitin addition promoted Mortierella growth and turnover in plant roots by fostering the development of cell walls and accelerating cell division, ultimately facilitating the production and transport of N, other nutrients (e.g., K⁺, and Ca²⁺), and energy for the growth of microbes and host plants.

6 Disentangling the effect of chitin addition on root-associated fungal communities in the plant-Mortirella system

By aligning high-quality reads to ITS and LSU databases using the MicroFisher workflow, we obtained 31 fungal genera across all samples and then performed microbial analyses at the genus level (Fig. 8). We found that chitin addition decreased the fungal alpha diversity (shown by the Shannon index), although the effect was not significant (Fig. 8A). Principal coordinate analysis (PCoA) and Permanova showed that chitin addition induced a significantly different fungal community composition (P < 0.05; Fig. 8B). These imply chitin addition, an organic N source, had a substantial impact on fungal communities in the plant root, which may play crucial roles in the growth, development, and fitness of host plants (White et al., 2019; Rana et al., 2020). Among 31 fungal genera detected, Neurospora, Fusarium, Mortierella, Peziza, Terfezia, and Linnemannia were the dominant fungal genera (relative abundance > 2%), accounting for over 90% of total reads on average across all samples (Fig. 8C). Not surprisingly, chitin addition significantly increased the relative abundance of Mortierella, consistent with the high expression of Mortierella genes associated with fungal growth and turnover (Fig. 7A). However, chitin addition significantly decreased the relative abundance of Peziza. Peziza is an ectomycorrhizal symbiont of Poplar, which can assist host plants with acquiring nutrients (Tedersoo et al., 2006). This implies plants may develop specific symbiotic strategies for their growth and fitness under different conditions. 

final report for SSARE_figures