Colletotrichum gloeosporioides, (Penz.) Penz. & Sacc.

Mehmood, Nasir, Yuan, Yuan, Ali, Mohammed, Ali, Muhammad, Iftikhar, Junaid, Cheng, Chunzhen, Lyu, Meiling & Wu, Binghua, 2021, Early transcriptional response of terpenoid metabolism to Colletotrichum gloeosporioides in a resistant wild strawberry Fragaria nilgerrensis, Phytochemistry (112590) 181, pp. 1-12 : 6-7

4.1. Plant materials, growth conditions, C. gloeosporioides treatment and tissue collection

The woodland strawberry accession F. nilgerrensis was used in this study. Runners derived from healthy plants were rooted in pots and incubated in the growth room (16 h light/8 h dark, 22–24 ◦ C day/26 ◦ C night cycle, 125 μmol m- 2 s- 1 light intensity, and 70% humidity) to obtain the healthy and vigorous plants.

The selected pathogenic fungus C. gloeosporioides was isolated by the Fruit Research Institute, Fujian Academy of Agriculture and maintained in Shaking Potato Dextrose (SPD) media. Fungi culture was incubated for about two weeks (10–14 days) in optimum temperature 28 ◦ C. To prepare conidiospores, the saturated cultures were passed out through double layer of cheese cloth. Conidial suspension was then transferred to centrifuge tubes (micro; 1.5) for centrifugation at 2500 rpm for 20 min. Supernatant of the centrifuged suspension was removed and discarded. The remaining pellet was further re-suspended and diluted to a final conidia concentration of 10 6 ml 1 with sterile distilled water containing 0.05% Tween-20 before using in plant spray treatment. The plants inoculated with the final conidia suspension of C. gloeosporioides were placed in culture chamber at temperature (26 ◦ C) up to 14 days for possible disease symptoms observation. Since the wild strawberry F. nilgerrensis was resistant to the fungi, no obvious disease symptom could be seen during the experimental time ( Figure S1 ), the fungal challenge was monitored later by transcriptome analysis of fungispecific-transcript detection ( Figure S2 ).

For sampling after the pathogen treatment, leaves from two-monthold runner-propagated healthy plants with unfolded green leaves at uniform size and color were inoculated with C. gloeosporioides conidia suspension (10 6 conidia per ml) in sterile distilled water containing 0.05% Tween-20. Three hours prior to pathogen treatment, the leaves were sprayed first with distilled water containing 0.05% of Tween-20, therefore, the time 0 h was considered as controls. The treated plants were incubated in the growth room at the same condition. All plants were watered daily by nutrition solution (1/4 MS medium). For RNASeq analysis, two biological replicates from treated leaves were sampled at 0 h, 3 h, 6 h, 12 h, 18 h, 24 h, 48 h and 72 h post infection (hpi) and handled. Each replicate consisted of nine tri-foliate leaves; each was collected from one individual plant. For qRT-PCR, three biological replicates were collected from the same time-points. Furthermore, another two biological replicates from fresh leaves at the indicated time points were collected for isolation of the phytochemical and volatile compounds. In total 60 plants were used for the treatments. All samples were immediately frozen in liquid nitrogen and then stored at 80 ◦ C until use.

Figures

Fig. 1. Determination of terpenoids in leaves challenged with C. gloeosporioides. (A) Total contents of monoterpenes and sesquiterpenes, (B–C) The contents of individual monoterpene and sesquiterpene identified. Data are average of two independent experiments.

Fig. 2. Global differentially-expressed-genes (DEG) in leaves of F. nilgerrensis after challenged by C. gloeosporioides. (A) DEG heatmap depicts clustering of the early upregulated genes (up to 18-h post challenge) and the late respondents (>24 h) in the upper and lower parts, respectively, with top GO terms indicated. (B) Major GO term enrichment in an early responsive gene cluster 7 showing that terpenoid biosynthesis genes are co-expressed with antipathogen immune-responsive pathway genes. Each time-point contains 2 biological replications and the expression data was zero-normalized before hierarchical clustering is performed (Pearson uncentered metric, average linkage ordering) using Cluster 3.0 version 1.58. Heatmap was examined and generated using Java Tree- View version 1.1.6r4. Gene Ontology (GO) enrichment analysis of the DEGs is implemented by the topGO R package-based Kolmogorov–Smirnov test.