Plant health status effects on arbuscular mycorrhizal fungi associated with *Lavandula angustifolia* and *Lavandula intermedia* infected by Phytoplasma in France

Arbuscular mycorrhizal fungi (AMF, Glomeromycota) form obligate symbioses with most land plants and provide ecosystem services, including improved nutrient availability, plant diversity, soil structure, and enhanced resistance to abiotic and biotic stresses. AMF have been shown to reduce or suppress plant disease symptoms caused by various phytopathogens, making them key players in sustainable cropping systems. Understanding the diversity and drivers of AMF communities in crop roots can inform strategies to leverage these fungi for plant health. Plant health status has been suggested to shape root-associated fungal communities; for example, AMF were more abundant and diverse in healthy Pisum sativum, and AMF diversity and composition shifted between healthy and diseased plants in Salix bioenergy plantations. However, these effects have not been evaluated under different agricultural systems. Organic practices often promote AMF colonization and soil AMF diversity (e.g., via crop rotation, cover crops, reduced synthetic inputs), while conventional high-input systems can negatively affect AMF, although findings are sometimes conflicting. This suggests interactive effects with other factors such as host plant health status. The objective of this study was to test whether AMF colonization, diversity, and community composition in roots of Lavandula angustifolia (lavender) and Lavandula intermedia (lavandin) are influenced by plant health status (asymptomatic healthy vs symptomatic diseased with stolbur phytoplasma ‘Candidatus Phytoplasma solani’) and whether these effects depend on agricultural practice (conventional vs organic) and plant species identity in fields in southeastern France.

Prior work indicates plant health status is related to root fungal community composition, with AMF often more abundant and diverse in healthy plants (e.g., Pisum sativum). Shifts in AMF diversity and composition between healthy and diseased hosts have been reported in Salix plantations. Agricultural management also influences AMF: organic systems generally enhance AMF colonization and spore abundance and maintain higher soil AMF diversity (e.g., via cover crops and absence of synthetic inputs), while conventional high-input systems and disturbances (e.g., flooding) can reduce AMF, although some studies show minimal effects. These mixed results suggest interactions with biotic factors such as host health status may modulate AMF communities.

Study system and sites: Four fields in southeastern France were selected, representing two agricultural practices (organic and conventional) for each of two species: Lavandula angustifolia (cv. Rapido, seed-derived) and Lavandula intermedia (cv. Grosso, vegetatively propagated). Fields were affected by stolbur phytoplasma (Candidatus Phytoplasma solani) with 20–30% symptomatic plants. Management and soil characteristics: For L. intermedia, the organic field (20 years organic; no fertilizer or pesticides; no tillage; plant cover of Fabaceae and Poaceae) had organic matter 3.20 ± 0.10%, total carbon 1.84 ± 0.01%, pH 8.25 ± 0.05. The conventional field (>20 years conventional; 30:30:40 kg NPK ha−1; herbicides and insecticides including propyzamid, cycloxydim, fluroxypyr or amidosulfuron, and chlorpyriphos-methyl; reduced tillage to 5–10 cm) had organic matter 2.70 ± 0.00%, total carbon 1.57 ± 0.01%, pH 8.25 ± 0.05. For L. angustifolia, the organic field (4 years organic; no fertilizer or pesticides; reduced tillage) had organic matter 5 ± 0.20%, total carbon 2.90 ± 0.09%, pH 8.2 ± 0.00, and the conventional field (>20 years conventional; 30:30:40 kg NPK ha−1; herbicides and insecticides; reduced tillage) had organic matter 4.2 ± 0.30%, total carbon 2.46 ± 0.18%, pH 8.25 ± 0.05. Plant health status: Healthy (asymptomatic) plants had no visible symptoms; diseased (symptomatic) plants showed typical phytoplasma symptoms (yellowing, reduced inflorescences), assessed by experts from CRIEPPAM. Sampling design: In July 2017, within a 20 m × 20 m area per field, 12 root samples were randomly collected from underground parts of 2‑year‑old healthy plants and 12 from diseased plants. Across factors (2 species × 2 practices × 2 health statuses × 12 replicates), 96 root samples were collected, rinsed, and used for colonization assessment and DNA extraction. Mycorrhizal colonization: Roots were cleared and stained (Phillips & Hayman, 1970). AMF colonization intensity (M%) was estimated by light microscopy using MYCOCALC. DNA extraction and sequencing: For each species, the 12 root samples per condition were pooled two by two, yielding six DNA samples. Dried, ground roots (100 mg) were extracted with FastPrep DNA Spin kit (MPBIO). AMF-specific primers FLR3/FLR4 targeting partial LSU were used, with 8-nt sample tags. PCR conditions followed prior work with annealing at 58 °C and elongation 30 s. Amplicons were pooled equimolarly and sequenced on Illumina MiSeq 150 bp paired-end (Fasteris, Geneva). Bioinformatics: Reads were processed with OBItools: assembly and filtering removed short (<100 nt) or rare (occurrence <2) reads, resulting in 138,593 unique sequences from 3,917,531 reads total. Unique sequences were clustered into MOTUs at 98% similarity using Sumatra and Markov Cluster algorithm (MCL). Taxonomic assignment of 3,260 MOTUs used ecotag against an AMF database generated from EMBL r136 via in silico PCR with FLR3/FLR4. Data processing in R 3.5.3 included aggregation of sequences by MOTU, removal of rare MOTUs (present in only one sample), outlier samples (Bray–Curtis scatter distance exceeding 95% CI), and MOTUs not assigned to AMF clades. Data were Hellinger-standardized (vegan::decostand). Final tables contained relative abundances of 190 MOTUs (lavender) and 180 MOTUs (lavandin). Statistics: Normality assessed by Shapiro–Wilk; variance by Levene. Depending on assumptions, Student’s t-test or Wilcoxon tests compared root colonization and diversity metrics between health statuses. AMF community distribution was evaluated by Multiple Factor Analysis (MFA). Effects of plant health status on colonization, diversity, and community structure were tested by PERMANOVA with Permutest. Dominant MOTUs (found in at least two-thirds of samples) were compared between healthy and diseased within each practice using chi-squared tests with adjusted standardized residuals. Analyses used R vegan.

AMF root colonization: In Lavandula angustifolia, colonization was significantly higher in healthy vs diseased plants in both practices: organic 27% (healthy) vs 15% (diseased); conventional 30% vs 5%. Plant health status explained ~30% of colonization variability in both practices (variance partitioning, Table S1). In Lavandula intermedia, colonization was also higher in healthy vs diseased: organic 42% vs 30%; conventional 12% vs 7%, with health status explaining ~15% of variability in each practice. AMF diversity: Rarefaction curves saturated. In L. angustifolia, health status affected AMF abundance and richness only under conventional practice, with higher abundance and greater richness in healthy plants; no effect under organic practice. In L. intermedia, health status did not affect AMF abundance or richness in either practice. AMF distribution (community structure): In L. angustifolia, MFA separated AMF communities by health status in both practices. MFA axis 1 explained 34.8% (organic) and 42% (conventional) of variance; axis 2 explained ≤16%. PERMANOVA indicated health status explained 16% (organic) and 19% (conventional) of variance in community distribution; residuals accounted for 84% and 81%, respectively. In L. intermedia, no significant effect of health status on community structure in either practice. Community composition and MOTUs: Roots were dominated by Glomeraceae, followed by unclassified Glomeromycetes, with smaller proportions of Claroideoglomeraceae, Acaulosporaceae, and Gigasporaceae varying by species, health, and practice. Among abundant MOTUs (present in ≥2/3 samples; sequences MN510817–MN510849), health status-related differences were observed. In L. angustifolia, 21 abundant MOTUs showed variable distributions by health and practice; e.g., under conventional practice, diseased roots had more Rhizophagus intraradices and Glomus sp2, while healthy had more Glomus sp5 and Glomus sp7; under organic, diseased had Glomus sp2, sp4, sp6, sp8; healthy had Glomus macrocarpum and Funneliformis mosseae. In L. intermedia, 23 abundant MOTUs varied with health and practice; under conventional, Rhizophagus irregularis was more abundant in diseased, and Glomus aggregatum, Glomus sp2, Rhizophagus intraradices in healthy; under organic, Glomus aggregatum and Rhizophagus sp5 were more abundant in diseased, Rhizophagus sp1 in healthy. Across species, four different MOTUs per species were significantly more abundant in healthy roots in either practice. Overall, plant health status increased colonization in both species, increased AMF abundance and richness only in L. angustifolia under conventional practice, and strongly structured communities in L. angustifolia but not in L. intermedia.

This study extends prior work by evaluating plant health status effects on root AMF communities under both organic and conventional management. Plant health status increased the intensity of AMF colonization in both lavender and lavandin, with a stronger effect in lavender (explaining ~30% of colonization variability) than lavandin (~15%), suggesting host identity influences symbiosis. Potential mechanisms include differences in root exudates between healthy and diseased plants and reduced carbon supply to AMF in phytoplasma-infected plants, both of which could modulate AMF establishment. Health status effects on AMF diversity were host- and management-specific: only lavender under conventional practice showed higher AMF abundance and richness in healthy plants, indicating interactive effects between plant health and agricultural management. Community structure analyses corroborated this pattern: health status significantly structured AMF communities in lavender across practices, while no significant structuring was detected in lavandin. Compositionally, health status differences were reflected more by shifts in MOTU abundances than by exclusive presence/absence, with several MOTUs (e.g., Funneliformis mosseae, Glomus macrocarpum) more abundant in healthy roots and known to alleviate disease or abiotic stress. These results suggest that plant health status, modulated by management and host identity, can shape AMF symbioses and select for potentially beneficial AMF taxa.

Plant health status is an important driver of root AMF colonization, diversity, and community composition in lavender cropping systems. Healthy plants exhibited higher AMF colonization in both lavender and lavandin, with stronger effects in lavender. In lavender, health status increased AMF abundance and richness and strongly structured communities, particularly under conventional management, whereas lavandin showed little to no diversity or structural response. Agricultural practices can modulate these effects, and host species identity is a key determinant. Abundant MOTUs enriched in healthy roots, including Funneliformis mosseae and Glomus macrocarpum, may contribute to protection against phytoplasma or other stresses and represent candidates for inoculum development. Future work should test causal roles of specific AMF taxa in conferring disease tolerance, explore underlying mechanisms (e.g., root exudation and carbon allocation), and assess generality across regions, cultivars, and management regimes.

The authors note that other unmeasured drivers (abiotic or biotic) likely influence AMF communities, as indicated by large residual variance in community analyses. Effects were assessed in two species at four fields within one region and season, which may limit generalizability. Community inferences are based on MOTU read abundances after pooling some root samples and bioinformatic filtering, which may underrepresent rare taxa and does not establish causality between specific AMF and plant health.