First Report of Gray and Pink <i>Botrytis cinerea</i> Causing Gray Mold on Blueberry Fruits ( <i>Vaccinium corymbosum</i> ) in Mexico

Mexico's 2023 blueberry production reached 11,400 hectares, with 96% destined for the United States (Aneberries, unpublish data). To meet U.S. import requirements, field growers report that Botrytis control during the final crop stages uses 10–30% of total management resources. In 2023 and 2024, 104 Botrytis strains were isolated from symptomatic blueberry fruit and flowers. Single spore isolates were obtained from samples collected from multiple cultivars under organic and conventional management in five major blueberry producing states: Jalisco, Michoacán, Baja California, Guanajuato, and Sinaloa. Most isolates initially were hyaline and gradually developed the characteristic gray pigmentation. One isolate (J029), however, exhibited an atypical phenotype, producing pink pigmentation as the colony matured. All strains were morphologically characterized, and molecular identification was conducted on three isolates, M015 (N 19°53´59.8", W 102°07´56.8"), S019 (N 24°45´48.24", W 107°37´27.68"), and J029 (N 20°45′58.3″, W 102°38′09.9″). On potato dextrose agar, all isolates formed gray mycelium after four days at 20 °C, except J029, which developed pink mycelium. After 20 days, several isolates produced black sclerotia. J029 formed randomly dispersed black sclerotia with pink margins and irregular, spherical or oblong/elliptic shapes. Sclerotia of the isolated Botrytis measured 0.4–7.8 × 0.4–6.0 mm (mean 2.3 ± 1.1 × 1.8 ± 0.8 mm, n = 50). The basal parts of the conidiophores were unbranched, septate, pigmented, and the apical part was hyaline and branched toward the apex with measurements 309-3215 x 6-23.4 µm (mean 1431 ± 503 × 13 ± 2.3 µm, n=25). Macroconidia were unicellular, hyaline, ovoid to ellipsoid, 5.2-19.3 × 3.9-13 µm (mean 10.3 ± 2.1 × 7.4 ± 1.3 µm, n=50) and were borne in grapelike clusters. Microconidia observed in J029 were unicellular, globose, hyaline, and measured 2.1–2.4 µm in diameter (n = 10); they emerged from phialides. The sequences of strain J029 were deposited in GenBank. The genes G3PDH, HSP60, RPB2 (accession numbers: PP942926, PP942927 and PP942929) showed 100%, 99.8% and 99.9% identity, respectively, with gene sequences reported for B. cinerea (KY364367.1, MN159921.1 and ON887325.1 ). To fulfill Koch’s postulates, the three isolates were evaluated on blueberry fruits of two varieties (Madeira and Maldiva). Ten fruits of each variety were disinfested and wound inoculated at the calyx end with 5 μl of a conidial suspension (1 × 106 conidia/ml). Ten wounded fruits were inoculated with sterile water as control. Fruits were incubated at 22°C for 13 days in a humid chamber. Experiments were repeated three times. Four days after inoculation, fruits developed gray mycelium on the calyx, while strain J029 produced a distinctive pink color. After one week, abundant sporulation and fruit rot were observed. By the second week, J029 formed black sclerotia with pink margins on the calyx. These symptoms are consistent with gray mold caused by B. cinerea, although pink pigmentation on the calyx is atypical and rarely reported for this species. No symptoms developed in control fruits. To our knowledge, this is the first official report of B. cinerea causing gray and pink mold and rot on blueberry fruits in Mexico, revealing a potentially overlooked, and diagnostically significant phenotype rarely seen in this genus. This pink pigmentation suggests possible bikaverin production (Plesken et al., 2021) and requires further study.

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