Characterization of Pestalotiopsis chamaeropis causing gray blight disease on tea leaves (Camellia sinensis) in Chongqing, China

Abstract

Gray blight, caused by Pestalotiopsis-like species, is one of the most serious diseases in tea [Camellia sinensis (L.) O. Kuntze] production. In this study, eight Pestalotiopsis isolates obtained from diseased tea leaves collected in Chongqing, China were identified as P. chamaeropis Maharachch, K.D. Hyde & Crous. Pestalotiopsis chamaeropis as a causal agent of tea gray blight disease was clearly distinguished from other species in the genus Pestalotiopsis by morphological characteristics and phylogenetic analysis based on a combination of the internal transcribed spacer (ITS), beta-tubulin (β-tubulin), and partial translation elongation factor 1-alpha (TEF) genes. Pathogenicity was confirmed by inoculation of a conidial suspension of P. chamaeropis on wounded tea leaves. Difenoconazole, a highly active and low toxicity fungicide, demonstrated significant antifungal effect towards P. chamaeropis, with an EC₅₀ of 0.470 µg/mL.

Résumé

La moisissure grise, causée par les espèces du genre Pestalotiopsis, est une des maladies les plus graves du thé (Camellia sinensis [L.] O. Kuntze). Dans cette étude, huit isolats de Pestalotiopsis ont été obtenus de feuilles de thé infectées collectées dans la municipalité autonome de Chongqing, en Chine, et ont été identifiés en tant que P. chamaeropis Maharachch., K. D. Hyde & Crous. Pestalotiopsis chamaeropis, en tant qu’agent causal de la moisissure grise du thé, a été nettement différencié des autres espèces du genre Pestalotiopsis par ses caractéristiques morphologiques et l’analyse phylogénétique basée sur la région de l’espaceur transcrit interne (ITS), sur le gène de la bêta tubuline (β-tubuline) et sur une partie du gène codant pour le facteur de la traduction de l’élongation 1-alpha (TEF). La pathogénicité a été confirmée en inoculant des feuilles de thé présentant des lésions avec une suspension conidiale de P. chamaeropis. Le difénoconazole, un fongicide très actif et de faible toxicité, a démontré, à une CE₅₀ de 0,470 µg/mL, un effet antifongique notable à l’égard de P. chamaeropis.

Keywords:

• difenoconazole
• morphological characteristics
• Pestalotiopsis chamaeropis
• phylogeny
• tea plant

Mots clés:

• caractéristiques morphologiques
• difénoconazole
• Pestalotiopsis chamaeropis
• phylogénie
• théier

Introduction

Gray blight of tea [Camellia sinensis (L.) O. Kuntze] is an important disease in terms of economics as it causes significant production loss and quality decreases in many commercial tea plantations in India (Joshi et al. 2009), Japan (Horikawa 1986), and China (Liu et al. 2017; Chen et al. 2017, 2018a, 2018b). Therefore, an understanding of the pathogens that cause this disease is a priority. For a long time, gray blight disease was thought to be caused mainly by Pestalotiopsis theae (Sawata) Steyaert (Jeewon et al. 2003; Keith et al. 2006; Hu et al. 2007; Maharachchikumbura et al. 2011, 2013a, 2013b). More recently, a combination of the study of the morphological characteristics (i.e., length and width of conidia, median cell length, colour of median cells, and number and length of the apical appendages) and the molecular phylogenies derived from multi-gene analysis of the internal transcribed spacer (ITS), beta-tubulin (β-tubulin) and partial translation elongation factor 1-alpha (TEF) genes has proven to be an effective for species identification, resulting in greatly improved the understanding of Pestalotiopsis taxonomy on C. sinensis (Wei et al. 2007; Maharachchikumbura et al. 2014, 2016; Liu et al. 2017; Chen et al. 2018a). Based on this approach, several studies identifying Pestalotiopsis-like species as the causal agent of gray blight disease on C. sinensis have shown that there is considerable species diversity in the genus Pestalotiopsis (Chen et al. 2017, 2018a, 2018b; Wang et al. 2019a, 2019b).

Pestalotiopsis species are common phytopathogens and can cause many types of diseases on a wide range of host plants, including hazelnut, bayberry, longan, apple, blueberry, chestnut, rambutan, grape and guava (Sun and Cao 1990; Sangchote et al. 1998; Xu et al. 1999; Keith et al. 2006; Joshi et al. 2009; Keith and Zee 2010; Crous et al. 2011; Chen et al. 2011; Maharachchikumbura et al. 2012, 2013a, 2013b; Evidente et al. 2012; Ren et al. 2013). Chemical fungicides as a standard commercial practice, have been considered effective tools for the management of various plant diseases. Given its low mammalian toxicity and high efficiency, difenoconazole is often recommended for the management of foliar crop diseases including anthracnose caused by Colletotrichum spp. (Baggio et al. 2018). However, the use of this fungicide has not been evaluated for the management of gray blight disease of tea plants. In our study, the characteristics of a new Pestalotiopsis species from symptomatic tissues of tea plants in Chongqing city of China was described based on its morphological characters and multi-gene phylogenies based on ITS, β-tubulin and TEF genes. Furthermore, the effect of difenoconazole as a potential fungicide active ingredient against the new Pestalotiopsis species was evaluated.

Materials and methods

Isolates from diseased tea leaves

More than 20 naturally infected leaves were collected from commercial tea plantations located in Ersheng, in the Chongqing district of China (N29ʹ38, E106ʹ52), in 2017. The surfaces of symptomatic tea leaves were washed with running tap water. Leaf pieces (4 × 4 mm) were taken from the margins of symptomatic tissues, and surface-sterilized by dipping in 0.1% HgCl₂ for 30 s, then in 75% ethanol for 30 s, and rinsed three times with sterile water. The surface-sterilized leaf pieces were transferred to potato dextrose agar (PDA) in Petri dishes and incubated in a growth chamber at 25 ± 2°C with 12 h light photoperiod. Two days after incubation, mycelia growing from the symptomatic tissues were transferred to fresh PDA by taking agar discs from the margins of colonies.

Morphological analysis

Pure cultures of the fungi were grown on PDA at 25 ± 2ºC and checked daily. Colony morphology was examined after incubation for 5–10 days and photographs of the upper and lower surfaces of all the colonies were taken on the sixth day. The shape, colour, cell number, and number of apical and basal appendages of conidia were determined using a U-TV0.5XC-3 microscope (Olympus, Japan). The size (length and width) of conidia and the length of apical and basal appendages were determined for 30 randomly selected conidia from a conidial suspension of each isolate that was prepared in sterile water.

DNA extraction, PCR amplification and sequencing

The total genomic DNA was extracted from fresh mycelium of each of eight isolates following a CTAB method described previously (Chen et al. 2018b). Fresh mycelium was scraped off the PDA surface after six days of growth. The partial gene sequences of the ITS, β-tubulin, and TEF genes were amplified with the universal primers ITS4/ITS5 (White et al. 1990), T1/Bt-2b (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997) and EF1-728F/EF-2 (O’Donnell et al. 1998;Carbone and Kohn 1999) (Supplementary Table 1). The PCR conditions for each of ITS, β-tubulin and TEF were as follows: an initial denaturing for 3 min at 95°C, followed by 38 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 56°C and elongation for 1 min at 72°C, with a final extension step for 10 min at 72°C. The amplification products were resolved in 1% agarose gels, and purified using a Gel Extraction Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions, then cloned into a pGW-T Vector (Tiangen, Beijing). The positive transformants were sequenced at the Shanghai Sangon Biotech Company (Shanghai, China).

Phylogenetic analyses

New obtained gene sequences were deposited in GenBank and the sequences of homologous Pestalotiopsis species were retrieved from GenBank for comparison (Table 1). Neopestalotiopsis clavispora CBS 447.73 was used as the outgroup. The gene sequences of the isolates were aligned with the representative sequences in the NCBI database using Clustal W (Tamura et al. 2013). Phylogenetic trees were built and viewed in Molecular Evolutionary Genetic Analysis (MEGA) software v. 7. Phylogenetic analyses of the gene sequences consisted of Maximum Parsimony (MP) and Neighbour-Joining (NJ) methods based on both the individual and combined ITS, β-tubulin and TEF gene sequences. To determine the confidence values for the grouping within a tree, a bootstrap analysis was performed using 1,000 resamplings of the data (Saitou and Nei 1987; Tamura et al. 2013). The names and accession numbers were recorded on phylogenetic trees.

Table 1. Fungal species isolated from tea plants and the reference isolates used for phylogenetic analyses

Species	Isolate*	Host	Location	Genebank accession number
				ITS	β-tubulin	TEF
Pestalotiopsis chamaeropis	LB5-1	Camellia sinensis	China	MH425898	MH425914	MH425906
	LB5-2	Camellia sinensis	China	MH425899	MH425915	MH425907
	LB11-1	Camellia sinensis	China	MH425900	MH425916	MH425908
	LB11-2	Camellia sinensis	China	MH425901	MH425917	MH425909
	LB11-3	Camellia sinensis	China	MH425902	MH425918	MH425910
	LB61-1	Camellia sinensis	China	MH425903	MH425919	MH425911
	LB61-2	Camellia sinensis	China	MH425904	MH425920	MH425912
	LB61-3	Camellia sinensis	China	MH425905	MH425921	MH425913
	CBS 186.71	Chamaerops humilis	Italy	KM199326	KM199391	KM199473
	CBS 237.38	–	Italy	KM199324	KM199392	KM199474
	CBS 113607	–	–	KM199325	KM199390	KM199472
	CBS 113604	–	–	KM199323	KM199389	KM199471
	LC3609	Camellia sp.	China	KX894989	KX895320	KX895206
	LC3619	Camellia sp.	China	KX894991	KX895322	KX895208
Pestalotiopsis hollandica	CBS 265.33	Sciadopitys verticillata	Netherlands	KM199328	KM199388	KM199481
Pestalotiopsis australis	CBS 114474	Protea susannae	South Africa	KM199334	KM199385	KM199477
	CBS 111 503	Protea susannae	South Africa	KM199331	KM199382	KM199557
Pestalotiopsis monochaeta	GL1735	–	–	KX642434	KX642435	KX642433
	CBS 440.83	Taxus baccata	Netherlands	KM199329	KM199387	KM199480
Pestalotiopsis lushanensis	LC8183	Camellia sp.	China	KY464137	KY464157	KY464147
	LC8182	Camellia sp.	China	KY464136	KY464156	KY464146
	LC4344	Camellia sp.	China	KX895005	KX895337	KX895223
Pestalotiopsis grevilleae	CBS 114127	Grevillea sp.	Australia	KM199300	KM199407	KM199504
Pestalotiopsis knightiae	CBS 114138	Knightia sp.	New Zealand	KM199310	KM199408	KM199497
	CBS 111963	Knightia sp.	New Zealand	KM199311	KM199406	KM199495
Pestalotiopsis brachiata	LC8189	Camellia sp.	China	KY464143	KY464163	KY464153
	LC2988	Camellia sp.	China	KX894933	KX895265	KX895150
Pestalotiopsis australasiae	CBS 114126	Knightia sp.	New Zealand	KM199297	KM199409	KM199499
	CBS 114141	Protea	Australia	KM199298	KM199410	KM199501
Pestalotiopsis kenyana	LC3633	Camellia sp.	China	KX894992	KX895323	KX895209
	LC6633	Camellia sinensis	China	KX895027	KX895360	KX895246
Pestalotiopsis telopeae	CBS 114161	Telopea sp.	Australia	KM199296	KM199403	KM199500
	CBS 114137	Telopea sp.	Australia	KM199301	KM199469	KM199559
Pestalotiopsis jinchanghensis	LC8190	Camellia sinensis	China	KY464144	KY464164	KY464154
	LC6636	Camellia sinensis	China	KX895028	KX895361	KX895247
Pestalotiopsis aggestorum	LC8187	Camellia sinensis	China	KY464141	KY464161	KY464151
	LC8186	Camellia sinensis	China	KY464140	KY464160	KY464150
Pestalotiopsis biciliata	CBS 236.38	Paeonia sp.	Italy	KM199309	KM199401	KM199506
	CBS 124463	Platanus x hispanica	Slovakia	KM199308	KM199399	KM199505
Pestalotiopsis diploclisia	CBS 115585	Diploclisia glaucescens	China	KM199315	KM199417	KM199483
	CBS 115449	Psychotria tutcheri	China	KM199314	KM199416	KM199485
Pestalotiopsis oryzae	CBS 353.69	Oryza sativa	Denmark	KM199299	KM199398	KM199496
	CBS 111522	Telopea sp.	USA	KM199294	KM199394	KM199493
Neopestalotiopsis clavispora	CBS 447.73	Decaying wood	Sri Lanka	KM199374	KM199443	KM199539

*The bold indicate isolates obtained in this study.

Pathogenicity tests

Pathogenicity tests for each isolate were conducted on healthy tea leaves of the common tea cultivar cv. Fudingdabai, which were surfaced-disinfected using 75% ethanol. For each isolate, 10 leaves were inoculated by the wound method that was described previously (Chen et al. 2018a, 2018b). Eight microlitres of a conidial suspension (1 × 10⁶ conidia/mL) was placed over each wound. Control tea leaves were inoculated with an equal volume of sterile water. All inoculated tea leaves were placed into Petri dishes with wet filter paper to maintain high humidity and then placed in a growth chamber at 25 ± 2°C until disease symptoms appeared. All fungal isolates included in the pathogenicity tests were re-isolated from the diseased tea leaves to confirm their identity by both molecular and morphological methods as described above. Experiments were repeated three times with similar results. The disease incidences (%) were calculated by counting the number of infected tea leaves over the total inoculated tea leaves. The diameter of the lesions developed on tea leaves was measured after six days of incubation.

Antifungal activity of difenoconazole

Difenoconazole (Syngenta, China) was evaluated for its antifungal activity based on its capacity to inhibit the mycelial growth of isolate LB61-3. The test fungicide was dissolved in water and used to amend PDA medium at concentrations of 0, 0.1, 1.0, 2.0, 4.0, and 6.0 µg difenoconazole/mL. A mycelial disk of 5-mm-diameter of fungal isolate LB61-3 taken from a 5-day-old culture was placed at the centre of the PDA medium. Mycelial disks on PDA without any test chemical served as controls. All the Petri dishes were incubated at 25 ± 2°C for 6 days. The percentage inhibition was calculated using a formula: percentage inhibition = [(d c-d t)/d c] ×100, where d c and d t are the mean colony diameters of the control and treatment sets, respectively. There were three replicates for each treatment. The effective concentration of the fungicide causing growth of inhibition of 50% (EC₅₀) was determined by probit-log analysis (Wang et al. 2016).

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics 20.0. Data were analysed by one-way variance (ANOVA) and means were compared according to Turkey’s HSD (p ≤ 0.05).

Results

Gray blight disease was observed in the commercial tea plantations of Chongqing, China, in 2017. The disease generally began with small brown spots. As the disease developed, the spots got larger and the colour of necrotic lesions changed to light gray at the late stage of the disease (Fig. 1a), ultimately leading to serious losses in tea production. These symptoms were similar to our previous study (Chen et al. 2018b).

Fig. 1 The morphological characteristics of Pestalotiopsis-like species isolated from tea plants. (a) Symptoms caused by gray blight disease on tea plants; (b) Cultural traits of Pestalotiopsis isolates on potato dextrose agar (PDA) medium at 25 ± 2°C for six days; (c) Conidial morphology of Pestalotiopsis isolates on PDA medium after 8 days incubation (scale bar = 10 um)

Morphological characteristics of Pestalotiopsis species

Eight isolates (LB5-1, LB5-2, LB11-1, LB11-2, LB11-3, LB61-1, LB61-2, and LB61-3) were collected from symptomatic tea leaves. All the isolates reached 65–75 mm diam. after 7 days incubation at 25 ± 2°C on PDA. Colonies on PDA were white, with moderate aerial mycelium on the surface and light yellow colour on the reverse, with an undulating margin (Fig. 1b). The conidiomata of all isolates forming on the surface were black and gathered closely, diffusing throughout the culture medium after 7–10 days incubation (Fig. 1b). Conidia were fusoid, straight to slightly curved, five celled, 4-septate, with septa darker than the rest of the cells, 19.7–28.4 (mean ± SD = 25.1 ± 3.2) μm in length and 6.1–9.3 (7.1 ± 1.1) μm in width; three median cells were doliiform to sub-cylindrical, concolorous, brown, but the two upper median cells were slightly darker than the lower median cell, with the length ranging from 13.7 to 18.1 (15.5 ± 1.6) μm; basal cells were hyaline, obconic with a truncate base; basal appendages arising from the apical crest were single, straight or slightly curved, tubular, and 4.6–8.5 μm long; the apical appendages were 2 to 4 (mostly 3), with the length ranging from 10.1 to 18.9 μm (Fig. 1c).

Phylogenetic analyses of the combined datasets

Phylogenetic analyses of 43 isolates in the genus Pestalotiopsis were performed using both MP and NJ methods on the basis of the single locus and concatenated datasets of the ITS, β-tubulin and TEF genes. Both methods produced similar results, thus only the MP tree is presented. As shown in Fig. 2, 43 isolates from C. sinensis and other host plants of Pestalotiopsis clustered in 16 clades, which represented 16 known species including P. chamaeropis, P. hollandica, P. australis, P. monochaeta, P. lushanensis, P. grevilleae, P. knightiae, P. brachiata, P. australasiae, P. kenyana, P. telopeae, P. jinchanghensis, P. aggestorum, P. biciliata, P. diploclisia and P. oryzae. The eight isolates (LB5-1, LB5-2, LB11-1, LB11-2, LB11-3, LB61-1, LB61-2, and LB61-3) in this study clustered with previously identified strains of P. chamaeropis with a high bootstrap support of 100%. BLAST searches indicated that the ITS, β-tubulin and TEF gene sequences of the isolates in this study shared 98–100% similarity with those from some other hosts published in GenBank in the same clade. Based on the molecular analysis and morphological characteristics, the eight isolates in this study were identified as Pestalotiopsis chamaeropis Maharachch, K.D. Hyde & Crous.

Fig. 2 Phylogenetic tree obtained using the MP method on the basis of combined ITS, β-tubulin and TEF gene sequence data from distinctive Pestalotiopsis species in this study (bold font) and published sequences. Neopestalotiopsis clavispora CBS 447.73 served as the outgroup. The numbers in the bootstrap test (1,000 replicates) are indicated above the branches and only values > 50% are shown

Pathogencity analysis

In pathogenicity tests of all the P. chamaeropis isolates, 40.0–76.7% of the inoculated tea leaves showed obvious disease symptoms (Table 2). Symptoms began as tiny brown or dark spots around the wound, two days after inoculation. These spots expanded to surrounding healthy tissues six days after inoculation (Fig. 3), with 3.0–6.5 mm diameter lesions (Table 2). Finally black acervuli were formed on the surface of the lesions. Although all of the P. chamaeropis isolates were pathogenic to tea leaves, they showed differences in virulence. Isolate LB61-3 was the most virulent producing typical lesions on infected leaves of 6.5 ± 0.3 mm diameter, while isolate LB5-1 was the least virulent with a lesion diameter of 3.0 ± 0.1 mm (Table 2). All the symptoms were identical to those observed in the field. The control tea leaves inoculated with sterile water did not show any symptoms throughout the duration of the experiments (Fig. 3). The fungi re-isolated from the lesions showed the same morphological characteristics and 100% sequence similarities with the ones used for the inoculations. The recovery frequency of P. chamaeropis from the inoculated lesions was 100%, while a recovery of 0% was recorded for the control treatment.

Table 2. Pathogenicity of the isolates of P. chamaeropis on the tea cv. Fudingdabai six days after inoculation

Species	Isolate	Infected tea leaves (%)^a	Lesion diameter (mm)^a
P. chamaeropis	LB5-1	40.0 ± 0.0d	3.0 ± 0.1c
	LB5-2	46.7 ± 3.3cd	3.6 ± 0.3c
	LB11-1	50.0 ± 5.8cd	5.0 ± 0.1b
	LB11-2	56.7 ± 3.3bcd	4.9 ± 0.3b
	LB11-3	60.0 ± 0.0abc	5.3 ± 0.2ab
	LB61-1	60.0 ± 5.8abc	5.8 ± 0.1ab
	LB61-2	70.0 ± 5.8ab	6.0 ± 0.3ab
	LB61-3	76.7 ± 3.3a	6.5 ± 0.3a

^aColumns with the same letter do not differ significantly per Tukey’s test (p ≤ 0.05).

Fig. 3 Typical symptoms caused by Pestalotiopsis chamaeropis on tea leaves

Effect of difenoconazole

Difenoconazole exhibited antifungal activity against P. chamaeropis which was dose-dependent in a wide range of concentrations from 0.1 to 6.0 µg/mL (Fig. 4). The inhibition percent ranged from 28.8% to 100.0%. Difenoconazole was very effective, with a 28.8% inhibition even at the low concentration of 0.1 µg/mL, and when the concentration reached 6.0 µg/mL, growth of the fungus was completely inhibited. The EC₅₀ of difenoconazole was 0.470 µg/mL.

Fig. 4 The antifungal activity of difenoconazole on mycelial growth rates of Pestalotiopsis chamaeropis.

Discussion

More than 20 Pestalotiopsis-like species have been thought to be associated with gray blight disease on C. sinensis. All the species belong to the three genera Pestalotiopsis, Neopestalotiopsis and Pseudopestalotiopsis, including P. longiseta, P. camelliae, P. furcate, N. clavispora, N. ellipsospora, P. menhaiensis, P. sichuanensis, P. kenyana, P. rhodomyrtus, P. chamaeropis, P. longiappendiculata, P. yanglingensis, P. trachicarpicola, P. lushanensis, P. jinchanghensis, Ps. camelliae-sinensis, Ps. ignota, Ps. cocos, Ps. indica, Ps. kubahensis, Ps. ampullacea, Ps. Chinensis, and Ps. theae (Takeda 2002; Joshi et al. 2009; Maharachchikumbura et al. 2014, 2016; Lateef et al. 2015; Liu et al. 2017; Chen et al. 2017, 2018a, 2018b; Wang et al. 2019a). Pseudopestalotiopsis and Neopestalotiopsis are two novel genera segregated from Pestalotiopsis, and in a previous study, five Pestalotiopsis-like species belonging to the three genera were found to be associated with tea gray blight disease in Chongqing city, China (Chen et al. 2017, 2018a, 2018b). In this study, eight P. chamaeropis isolates from diseased tea leaves were identified, and this is the first description of the characteristics of P. chamaeropis causing gray blight disease on C. sinensis in Chongqing city. The shape and size of the conidia of P. chamaeropis have some differences with the other four species, and the most important differences are with respect to the median cells and apical appendages of the conidia. Three median cells of the conidia of P. chamaeropis are concolourous, which can be easily distinguished from N. clavispora (Maharachchikumbura et al. 2014; Chen et al. 2018a, 2018b). The numbers and lengths of apical appendages of P. chamaeropis range from two to four (mostly three) and 10.1 to 18.9 μm, while P. camelliae has three to seven apical appendages, with the length ranging from 13.5 to 32.4 μm (Chen et al. 2017, 2018a, 2018b). Although P. chamaeropis has some distinct morphological differences with other species, a combination of morphological evaluation with an analysis of the ITS, β-tubulin and TEF gene regions provides the best resolution for distinguishing closely related species.

All the eight P. chamaeropis isolates used in this study were shown to be pathogenic to wounded tea plants; in contrast, symptoms were not usually observed on unwounded leaves (data not shown), suggesting that wounding may be necessary for symptom development. Similar results also have been confirmed in previous studies (Chen et al. 2018b; Wang et al. 2019a). Pestalotiopsis chamaeropis, initially isolated from the diseased leaves of dwarf palm (Chamaerops humilis), has also been identified as the pathogen-causing necrotic spots on mint-bush, Prostanthera rotundifolia, in Australia (Maharachchikumbura et al. 2014) and Camellia sp. in Jiangxi, China (Liu et al. 2017). The virulence of the eight P. chamaeropis isolates varied to some extent, which was consistent with the results that different Pestalotiopsis-like isolates within the same species showed variability in pathogenicity (Chen et al. 2018b; Wang et al. 2019a, 2019b). This indicates that pathogenicity may be isolate dependent rather than species dependent. Further research is needed to understand the basis for the differences in virulence among the isolates within the same species or species within the same genus. Isolate LB61-3, which showed stable and strong pathogenicity on tea plants, was used to test the antifungal activity of difenoconazole and the results showed that difenoconazole was effective against P. chamaeropis. Fungicides are the most important and commonly method used to control gray blight. To date, however, this fungicide has not been applied for the management of tea gray blight. Difenoconazole has been proven a useful fungicide for the management of chilli anthracnose caused by Colletotrichum acutatum (Gao et al. 2017), although it has a lower EC₅₀ value (0.22 μg/mL) against this fungus than for inhibiting growth of P. chamaeropis. The efficacy of fungicides and the sensitivity of the pathogen to those fungicides are important knowledge for effective disease management in the field.

Supplementary material

Supplemental data for this article can be accessed online here: https://doi.org/10.1080/07060661.2020.1816582.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) [31700607]; Fundamental Research Funds for the Central Universities [XDJK2020B046]; and Innovation and Entrepreneurship Training Program for College Students [5240101298 and 5240101234] for financial support.