As a perennial woody plant, the rubber tree (Hevea brasiliensis) must adapt to various environmental challenges through gene expression in multiple cell types. It is still unclear how genes in this species are expressed at the cellular level and the precise mechanisms by which cells respond transcriptionally to environmental stimuli, especially in the case of pathogen infection. Here, we characterized the transcriptomes in Hevea leaves during early powdery mildew infection using single‐cell RNA sequencing. We identified 10 cell types and constructed the first single‐cell atlas of Hevea leaves. Distinct gene expression patterns of the cell clusters were observed under powdery mildew infection, which was especially significant in the epidermal cells. Most of the genes involved in host–pathogen interactions in epidermal cells exhibited a pattern of dramatically increased expression with increasing pseudotime. Interestingly, we found that the HbCNL2 gene, encoding a nucleotide‐binding leucine‐rich repeat protein, positively modulated the defence of rubber leaves against powdery mildew. Overexpression of the HbCNL2 gene triggered a typical cell death phenotype in tobacco leaves and a higher level of reactive oxygen species in the protoplasts of Hevea leaves. The HbCNL2 protein was located in the cytomembrane and nucleus, and its leucine‐rich repeat domain interacted with the histidine kinase‐like ATPase domain of the molecular chaperone HbHSP90 in the nucleus. Collectively, our results provide the first observation of the cellular and molecular responses of Hevea leaves to biotrophic pathogen infection and can guide the identification of disease‐resistance genes in this important tree species.

Stem-end rot (SER) caused by Lasiodiplodia theobromae is one of the most critical diseases of mango in China. The demethylation inhibitor fungicide prochloraz has been widely used in China to control mango diseases. Isolates (n = 139) of L. theobromae were collected in 2019 from six mango-producing regions in Hainan Province, China. The fungicide sensitivity of L. theobromae isolates to prochloraz revealed that the EC50 values ranged from 0.0006 to 16.4131 µg/ml. A total of 21 of the 139 isolates were categorized as resistant to prochloraz. The resistant isolates sprayed with prochloraz could not be effectively controlled in detached fruits. The mycelial growth, conidia germination and ability to grow at temperatures ranging from 12–35℃ of resistant isolates decreased, suggesting fitness penalties . The experiment showed that after treatment with 10 µg/ml prochloraz, the content of ergosterol in the mycelia of the sensitive isolate decreased by 80.23%, whereas the resistant strain decreased by only 57.52%. The damages of membranes in the sensitive isolates were more serious than for resistant isolates. The target gene CYP51 and the ATP-binding cassette subfamily ABCG gene were cloned, but no mutation was found. When treated with prochloraz, the expression of CYP51 and ABCG in resistant isolates was significantly higher than those in the sensitive isolates. Thus, induced expression of its target gene combined with the induction of expression drug efflux transporters appeared to mediate the prochloraz resistance of L. theobromae.

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.

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