Xylem Sap Mycobiota in Grapevine Naturally Infected with Xylella fastidiosa: A Case Study: Interaction of Xylella fastidiosa with Sclerotinia sclerotiorum

Abstract

Grapevine (Vitis vinifera) is a key crop in Mediterranean agriculture, now increasingly threatened by Xylella fastidiosa subsp. Fastidiosa (Xff), the causal agent of Pierce's disease. This study investigated: (1) the diversity of culturable fungal endophytes in the xylem sap of naturally Xff-infected grapevines, and (2) the interaction between Xff and the pathogenic fungus Sclerotinia sclerotiorum identified in the sap. The xylem sap was collected from Cabernet Sauvignon vines in Mallorca, Spain, and fungal communities were characterized using culture-dependent methods. Both beneficial fungi (e.g., Aureobasidium pullulans, Rhodotorula mucilaginosa) and pathogenic species (e.g., S. sclerotiorum, Cladosporium sp., Alternaria alternata, and the Phoma complex) were isolated from both Xff-positive and Xff-negative plants, indicating similar community profiles. Although limited by small sample size, these findings offer preliminary evidence of complex ecological interactions between Xff and the xylem-associated mycobiota, with potential implications for grapevine health and disease development under varying environmental and management conditions. Further experiments under controlled conditions revealed that grapevines co-inoculated with Xff and S. sclerotiorum showed increased disease severity, suggesting a synergistic interaction. These preliminary results highlight the complex interplay between Xff and the fungal endophytic microbiome, which may modulate grapevine susceptibility depending on environmental and management conditions.

Keywords: Sclerotinia sclerotiorum; Xylella fastidiosa; cross-kingdom interactions; endophytic mycobiota; grapevine; synergism.

Figure 1

Mycobiota diversity in xylem sap collected from a vineyard in Mallorca, Spain. Data from 86 fungi isolates, obtained from two plants that tested positive and three plants that tested negative for the occurrence of Xff. No identification: fungal genus unclassified and referred to as unidentified taxon.

Figure 2

Symptoms of S. sclerotiorum in Cabernet Sauvignon grapevines at 7 dpi. (A) Broken stems from the point of infection because of the injury. Necrotic lesions (arrows), brown in color, with darker edges extended along the stem that remained green, compromising petioles that were totally or partially necrotic and therefore led to the wilting of the leaves in the affected shoot (B) detail of necrotic petiole and leaf wilting.

Figure 3

Symptoms of Ss in C. Sauvignon grapevine at 7 dpi (A) Epinasty and necrosis in the stem with total or partial decay of the shoot or its leaves. (B) Detail of the necrotic lesion in a young shoot fractured due to spontaneous breaking. (C) Epinasty and distal wilting in terminal shoot’s leaves.

Figure 4

Symptoms on grapevine plants control (Mock), infected with Sclerotinia (Ss), Xylella (Xff) and the combination (Ss + Xff). (A) Number of necrotic lesions on stems; (B) necrotic stem length and (C) number of necrotic petioles per grapevine plant analyzed. Measurements of symptoms were performed at 7 dpi. The bars represent the average of each measure. The experiment followed a completely randomized design with four replicates per treatment. Statistical analysis using one-way ANOVA revealed a significant effect: In (A): F = 5.65, p = 0.0119, df = 15; (B): F = 29.67, p ≤ 0.0001, df = 15; (C): F = 68.28, p < 0.0001, df = 15. Different letters indicate statistically significant differences (p < 0.05).

Figure 5

Principal component analysis (PCA) biplot showing the distribution of treatments and disease severity variables in infected and mock-inoculated plants. Plants control (Mock), infected with S. sclerotiorum (Ss), X. fastidiosa (Xff) and the combination (Ss + Xff).

Figure 6

Area Under the Disease Progress Curve (AUDPC) for necrotic stem length in two treatments. Disease progression was assessed at 0-, 7-, and 10-days post inoculation (dpi) and quantified as necrotic stem length (cm). Panel (A) represents the AUDPC for plants inoculated with Ss, and Panel (B) corresponds to plants inoculated with Xylella fastidiosa subsp. fastidiosa + Ss (Xff-Ss). AUDPC values were calculated using data from four biological replicates per treatment. The Xff-Ss treatment exhibited a significantly higher disease severity, with an average AUDPC value of 523.56 ± 278.64, compared to 155.96 ± 83.31 in the Ss treatment (mean ± standard error). Statistical analysis using one-way ANOVA revealed a significant difference between treatments (F = 11.54, p = 0.0008, df = 15). These results suggest a synergistic or enhancing effect of Xff on disease development when co-inoculated with Ss.

Figure 7

Symptoms on Cabernet Sauvignon grapevine plants at 7 dpi in Mock, Ss, Xff and Ss + Xff. Details of grape bunches and stem necrosis, stem breakage, wilting and falling of leaf petioles caused by Ss in plants inoculated with Xff (below).

Figure 8

Stomatal conductance and chlorophyll content in grapevine leaves across different treatments. Physiological parameters were measured at 10 days post inoculation in grapevine plants subjected to four treatments: Control (Mock), Xylella fastidiosa subsp. fastidiosa (Xff), Sclerotinia sclerotiorum (Ss), and co-inoculation with X. fastidiosa + S. sclerotiorum (Xff/Ss). Chlorophyll content (SPAD units) differed significantly among treatments (F = 2.01, p = 0.1664, df = 15). The highest mean value was observed in Xff/Ss (12.82 ± 4.69), followed by Xff (10.19 ± 3.01), Mock (9.26 ± 1.63), and Ss (7.73 ± 1.30). Tukey’s test indicated that Xff/Ss was significantly different from Ss (p < 0.05). Stomatal conductance (mmol m⁻² s⁻¹) also varied significantly among treatments (F = 90.69, p < 0.0001, df = 15). The Ss treatment exhibited the highest conductance (451.1 ± 20.1), significantly higher than Xff/Ss (362.3 ± 8.1), Mock (199.4 ± 36.3), and Xff (139.0 ± 42.7), with all pairwise differences significant (p < 0.05). Bars represent the mean ± standard deviation of four biological replicates per treatment. Different letters indicate statistically significant differences according to Tukey’s HSD test (p < 0.05).