First report of fludioxonil resistance isolate of Colletotrichum fructicola emerging on strawberry in Japan

doi:10.21203/rs.3.rs-3863817/v1

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First report of fludioxonil resistance isolate of Colletotrichum fructicola emerging on strawberry in Japan

https://doi.org/10.21203/rs.3.rs-3863817/v1

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published 16 Apr, 2024

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The species composition of the strawberry anthracnose pathogen population in Saga Prefecture was investigated through phylogenetic analysis of the nucleotide sequences of the intergenic regions of the apn2 and MAT1-2-1genes. Here, 77 out of the 80 Colletotricum isolates used were classified as C. fructicola, and the remaining three were classified as C. siamense. The 77 C. fructicola isolates were tested for sensitivityto fludioxonil, which is widely used as the main fungicide in rotational programs in Saga Prefecture. High levels of resistance to fludioxonil were observed in 14 C. fructicola isolates tested for sensitivity to mycelial growth in fludioxonil-containing media. Furthermore, two inoculation assays using strawberry seedlings treated with fludioxonil were performed on five of the 14 isolates that showed high levels of resistance. The effectiveness of fludioxonil was significantly low in these isolates. To our knowledge, this is the first study to report fludioxonil resistance in a C. fructicola population.

Anthracnose

Colletotrichum fructicola

fludioxonil

Strawberry

Resistance

Colletotrichum spp. are fungal plant pathogens that cause severe disease in fruits and vegetables. C. acutatum and C. gloeosporioides species complexes (CGSC) are the major causal agents of anthracnose in fruits such as apples, peaches, grapes, blueberries, and strawberries (Dowling et al. 2020) in temperate climate regions. Strawberry anthracnose is the most prevalent in Japan. It damages fruits, crowns, runners, petioles, and stipules.

Saga Prefecture (north latitude 32° 57 − 33° 37, east latitude 129° 44–130° 32) is in the northern part of the Kyushu region, which is one of the main strawberry-producing areas in Japan. Strawberry anthracnose is mainly transmitted through conidial dispersal via rain splashes. Anthracnose-induced fruit rot caused by infection with rain-splashed conidia is a major challenge in strawberry production worldwide (MacKenzie and Peres 2012). This fruit damage is avoidable in the northern part of the Kyushu region, including Saga Prefecture, as strawberries in this area are cultivated in greenhouses from November to May to protect them from rain-splashed conidial infection. The most adverse effect of anthracnose in this area is damping off disease in stocks, which occurs from the seedling-raising stage (from April to September) to immediately after planting in the field (Inada et al. 2005, 2008). Rain-protected cultivation in vinyl houses is recommended to prevent rain-splash conidial infections and subsequently control damping off (Hirayama et al. 2008, 2010; Inada et al. 2005, 2009, 2011). However, rain-protected cultivation is not common in the northern part of the Kyushu region because the seedling-raising period occurs during the typhoon season. Typhoons often damage vinyl houses, making farmers unwilling to use this cultivation method. Therefore, the control of damping off solely relies on fungicide application in the northern part of this region, especially in Saga Prefecture. Rotational application of multiple fungicides, including propineb, copper oxychloride, mancozeb, thiophanate-methyl, and QOI, is employed as a control strategy. However, reduced control efficacy against strawberry anthracnose has been observed for thiophanate-methyl (Adachi & Watanabe, 2014; Hirayama et al., 2008; Inada et al., 2009; Kikuchi et al., 2010) and QOI (Adachi and Watanabe 2014; Hirayama et al. 2008; Inada et al. 2009, 2010; Kikuchi et al. 2010; Yonezawa 2010) in various parts of Japan. Thus, fludioxonil has been used as an alternative to thiophanate-methyl and QOI (Inada et al. 2009) since 2006 in Saga Prefecture. Fludioxonil has a high level of efficacy that can be retained even after rainfall (Takeyama et al. 2022) against strawberry anthracnose (Adachi and Watanabe 2014). Owing to its ability to control rain splash dispersal, fludioxonil is widely used as the main fungicide in rotational programs in Saga Prefecture. However, this heavy usage has increased the risk of resistance to fludioxonil in the strawberry anthracnose pathogen population in the area.

The objective of this study was to assess the risk of fludioxonil resistance in strawberry anthracnose pathogen populations in Saga Prefecture. (i) Our examination of the species composition of the pathogen population revealed that C. fructicola, a member of the CGSC, dominated the Saga Prefecture. (ii) We performed a sensitivity test for mycelial growth in fludioxonil-containing media on C. fructicola isolates, the major causal agent of strawberry anthracnose in Saga Prefecture. (iii) We further evaluated the efficacy of fludioxonil in controlling C. fructicola isolates using an inoculation assay on strawberry seedlings treated with fludioxonil.

Sample collection

Diseased seedlings were collected from 32 strawberry fields across Saga Prefecture from August to October 2019. Colletotrichum isolates were collected from the crowns of seedlings. The crown was surface-sterilized in 2% antiformin aqueous solution for 1 min, washed with tap water for 15 min, and dried on a sterilized paper towel. Tissue samples were sliced from the crown, placed on a water ager plate, and incubated for 2–3 days at 25°C. Pure cultures were obtained through single hyphal or conidial isolation from the mycelia growing on tissue slices. Alternatively, Colletotrichum isolates were collected via single conidial isolation from a conidial mass formed on the leaf petiole lesions of the seedlings. We collected 1–6 Colletotrichum isolates from each field, resulting in a total of 80 isolates (Table S1, Fig.S1). The strawberry anthracnose-causing standard C. fructicola strains 96C-1 and 03-33-1 used in the present study were collected in Kanzaki City in 1996 and Karatsu City in 2003, respectively, in Saga Prefecture (Inada et al. 2008).

ApMat sequence analysis

Total DNA was extracted from the mycelia on PDA medium as previously described (Yokosawa et al. 2017) and used as a template for PCR amplification of the ApMat region. PCR was performed using AM-F and AM-R (Silva et al. 2012) as primers. KOD FX Neo (Toyobo, Osaka, Japan) was used as the enzyme for PCR according to the manufacturer’s instructions. The PCR conditions were as follows: 30 cycles of heat denaturation at 94°C for 45 s, annealing at 60°C for 45 s, extension reaction at 72°C for 1 min, followed by heat denaturation at 94°C for 3 min and extension reaction at 72°C for 1 min. The resulting PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio Inc., Shiga, Japan) and sequenced directly using AM-F and AM-R (Silva et al. 2012) as sequencing primers. Phylogenetic analysis was performed for the resulting ApMat sequences using MEGA version 11 (Tamura et al. 2021). ApMat sequences from 52 strains (including 36 type strains) of 29 Colletotrichum species selected by Yokosawa et al. (2017) were used as reference sequences for CGSC in the phylogenetic analysis (Fig. S1). Multiple sequence alignments were generated using the MUSCLE program in MEGA version 11 (Tamura et al. 2021). Genetic distances between sequences were calculated using Kimura's two-parameter (Kimura 1980), and a phylogenetic tree was constructed using the neighbor-joining method. The reliability of the phylogenetic tree was calculated using a bootstrap test with 1000 replicates. The ApMat sequences of the 80 field isolates were registered in the DDBJ, EMBL, and GenBank nucleotide sequence databases (accession numbers LC769252 to LC769331).

Sensitivity assay for mycelial growth to fludioxonil

Mycelial disks (4 mm in diameter) of each tested isolate were punched out of the margin of 7-day-old colonies on a PDA plate and incubated on PDA plates containing 0.1, 1, 10, 100, and 1000 mg/mL fludioxonil at 25°C for 4 days. The diameters of the colonies grown on the PDA plates were measured and compared with those grown on plates without fludioxonil to calculate the mycelial growth rate. The sensitivity assay for mycelial growth was repeated twice for each combination of the isolates and fludioxonil. The mycelial growth rate was calculated as the mean value of the two experiments.

Inoculation test for evaluating the control efficacy of fludioxonil

Strawberry seedlings (variety Saga i9) were grown in vinyl pots with a diameter of 9 cm. When three to four true leaves had completely expanded, strawberry seedlings were sprayed with 15 mL fludioxonil wettable powder solution per plant at a practical concentration (200 ppm) using a hand spray. The culture was shaken for 7 days at 25°C in a potato dextrose liquid medium, adjusted to 1.0×10⁵ conidia/mL, and sprayed on five seedlings (7 mL/plant) 4 h after fludioxonil application to prepare the conidial suspension of each tested isolate. The inoculated seedlings were placed in plastic bags and maintained in a humid chamber at 28°C for 72 h. Strawberry seedlings were kept in a glass chamber at approximately 25–35°C following removal from the bag. Symptoms were observed in all inoculated seedlings 13 days after inoculation. The degree of disease was evaluated in each inoculated seedling using the following index:

1: No more than 5 small lesions are observed on the leaves

2: 5–25 small lesions are observed on the leaves, or

one petiole is broken.

3: 26 or more lesions are observed on the leaves, or two petioles are broken.

4: Stock is dead

The degree of disease was also evaluated based on symptoms on the leaves of the inoculated seedlings using the following index:

1: No more than 5 small lesions are observed on the leaves

2: 5– 25 small lesions are observed on the leaves

3: 26 or more lesions are observed on the leaves

Disease severity was calculated using the following formula:

$$Disease severity=\sum \begin{array}{c}\left(\frac{degree of disease}{4 \times number of strains surveyed}\right) \times 100\end{array}$$

Species composition in the strawberry anthracnose pathogen population in Saga Prefecture

ApMat sequences from 80 field isolates collected in the present study were aligned with 52 reference sequences selected by Yokosawa et al. (2017). In the multiple alignment sequences, two types of ApMat sequences with lengths of 712b and 714b were obtained from field isolates and assigned as ApMat genotypes A and B, respectively. Among the 80 field isolates, 77 contained A-type sequences (Table S1, Fig. S1). Only three field isolates collected from Ogi City possessed type B sequences (Table S1, Fig. S1). Alignment with reference sequences revealed that the A-type sequence shared 100% homology with LC217783 from C. fructicola. In the phylogenetic tree (Fig. S2), the A-type sequence and LC217783 formed a single cluster with the other two reference sequences from C. fructicola (JQ807838 and JQ807839), which were obtained from the type strains of this species. The 77 isolates possessing the A-type sequence were identified as C. fructicola (Table S1). The B-type sequence exhibited the highest homology (98%) with JX145313, JQ899289, JQ807842, and KJ155453 among the 52 reference sequences sourced from CGSC. These four reference sequences were obtained from type strains of C. siamense. In the phylogenetic tree, the B-type sequence formed a single cluster with these four reference sequences and the other two sequences (JQ894582 and JQ807841) from the type strains of C. siamense (Fig. S2). Consequently, the three isolates with B-type sequences were identified as C. siamense. (Table S1).

Sensitivity of mycelia to fludioxonil

The 77 field isolates of C. fructicola were tested for the sensitivity of their mycelia to fludioxonil. The sensitivities of the tested field isolates were clearly categorized into types I and II based on the mycelial growth rates at 0.1 mg/mL and 1 mg/mL fludioxonil (Table S2, Fig. 1). Fourteen isolates grew at rates of > 75% at 0.1 mg/mL or 1 mg/mL fludioxonil (Table S2, Fig. 1) and were categorized as type I. In contrast, the remaining 63 isolates were more sensitive to fludioxonil at both concentrations. Their growth rates were < 70% at 0.1 mg/mL and < 50% at 1 mg/mL fludioxonil, and they were categorized as type II (Table S2). There was a statistically significant difference in the sensitivity of mycelia between type I and II isolates at both fludioxonil concentrations (t-test, 95% level; data not shown). Although no statistically significant differences in mycelial growth rates were observed between the two groups at fludioxonil concentrations > 10 mg/mL, type I isolates exhibited higher growth rates than type II isolates (Table S2 and Fig. 1). Fourteen type I isolates were collected from 10 fields, most of which were in Karatsu and Genkai cities in the northwestern part of Saga Prefecture (Table S2, Fig. S1).

Mycelial sensitivity to fludioxonil in the standard C. fructicola strains 03-33-01 and 96C-1 (Inada et al. 2008) was also evaluated. Mycelial growth was inhibited to the same level as that of type II isolates at all fludioxonil concentrations (Table S2); that is, the two standard C. fructicola strains were categorized as type II.

Control efficacy of fludioxonil against type I isolates

An inoculation assay was performed on strawberry seedlings treated with fludioxonil to further confirm the differences in fludioxonil sensitivity between type I and II isolates. Four field isolates, namely 1915-02, 1916-03, 1924-01, and 1924-02, were used to represent type I isolates, whereas standard C. fructicola strains 03-33-01 and 96C-1 (Inada et al. 2008) represented type II isolates in the first trial. Disease severity was measured by observing symptoms on the entire seedling, and 35–10 disease severities were evaluated following the inoculation of the four type I isolates (Table 1). The control efficacy of fludioxonil against the four field isolates ranged from 0 to 50 (Table 1). In contrast, fludioxonil sustained high efficacy on seedlings inoculated with type II standard C. fructicola strains (Table 1). No symptoms were observed in the seedlings, and the resulting efficacy was approximately 100 against the two standard strains (Table 1). To estimate the control efficacy more precisely, symptoms on the leaves of inoculated seedlings were only incorporated during the measurement of disease severity in the second trial. Three field isolates (1915-02, 1916-03, and 1924-02) from the first trial and 1915-03 were used as representative type I isolates, whereas 1919-02 represented type II field isolates in the second trial. The efficacy of fludioxonil against type I isolates was considerably reduced in the second trial. Disease severity evaluated after the inoculation of the four type I isolates ranged from 30 to 67.5, and the resulting fludioxonil efficacy ranged from 27.5 to 57.5 (Table 1). In contrast, no symptoms were observed on the leaves or the entire inoculated seedlings following inoculation with Strain 1919-02. Similarly, the resulting efficacy was 100 against this type II isolate in the first trial (Table 1). The efficacy of fludioxonil against type I isolates was markedly reduced in both trials. Therefore, we concluded that the four type I isolates tested were fludioxonil-resistant.

Table 1

Control efficacy of fludioxonil against Colletotrichum fructicola isolates collected from strawberry in Saga Prefecture
Category of^a sensitivity to fludioxonil	Isolate	Disease severity^b
		First trial		Second trial
		Symptom on entire seedling		Symptom on leaf
		Fludioxonil	Water control	Fludioxonil	Water control
Type I
	1915-02	10 (50)	20	67.5 (0)	57.5
	1915-03	-	-	30 (0)	27.5
	1916-03	35 (0)	35	32.5 (0)	27.5
	1924-01	30 (0)	30	-	-
	1924-02	25 (29)	35	40 (24)	52.5
Type II
	1919-02	-	-	0 (100)	40
	03-33-01	0 (100)	45	-	-
	96C-1	0 (100)	20	-	-
^a Type I and Type II were categorized in terms of the differences in mycelial growth rates at 0.1 and 1mg/mL fluodioxonil. Type I, more than 75% of mycelial growth rat at 0.1mg/mL or 1 mg/mL fludioxonil, respectively; type II, lower than 70% of mycelial growth rate at 0.1mg/mL and lower than 50% of mycelial growth rate at 1 mg/mL fludioxonil, respectively. These two types correspond to those in Fig. 1.
^b Control efficacies are shown in parentheses.

Sequence information from the ApMat region was used to investigate the species composition of strawberry anthracnose pathogens in the present study. The ApMat region can provide sufficient phylogenetic information to resolve species in CGSC (Jayawardena et al. 2016; Liu et al. 2015; Sharma et al. 2013, 2015; Silva et al. 2012). The 80 Colletotrichum isolates used in the present study were collected from strawberry fields in 13 cities covering almost all strawberry production areas in the Saga Prefecture. Therefore, they are considered representative of the strawberry pathogen population in this prefecture. Phylogenetic analysis inferred from ApMat sequence information revealed that most field isolates (77 of 80) were C. fructicola. Therefore, it can be concluded that C. fructicola is the predominant cause of strawberry anthracnose in Saga Prefecture. C. fructicola remains the most commonly examined species in the anthracnose pathogen populations on strawberries in Japan to date (Egashira et al. 2016; Ibaraki Agricultural Center 2020; Shirasu 2018). The simple species composition of C. fructicola may be a general feature of the population structure of strawberry anthracnose pathogens in Japan. Three of the 80 isolates were classified as C. siamense, indicating that C. siamense was a minor component of the pathogen population. The three C. siamense isolates were distributed across a single field in Ogi City, Saga Prefecture. In light of its minority and localization, it is reasonable to assume that damage from strawberry anthracnose caused by C. siamense, if any, is limited or can be ignored in strawberry production in this Prefecture.

In the present study, we examined C. fructicola as the major causal agent of strawberry anthracnose to assess the risk of fludioxonil resistance in a pathogen population in Saga Prefecture. The 77 C. fructicola field isolates were initially tested for the sensitivity of their mycelia to fludioxonil. Here, 14 isolates exhibited significantly higher growth rates than the other 63 isolates tested at 0.1 mg/mL and 1 mg/mL fludioxonil concentrations. The 14 and 63 field isolates were subsequently categorized as types I and II, respectively. A clear difference in sensitivity was observed between the two types in the inoculation assay of strawberry seedlings treated with fludioxonil. Efficacy was substantially reduced against the five types of category I isolates used in the assay. This reduction sharply contrasts with the high levels of efficacy against type II representatives. The reduced inhibitory effect on mycelial growth and efficacy indicated that at least five type I isolates used in the inoculation assay were fludioxonil-resistant. Fludioxonil belongs to the phenylpyrrole family of fungicides derived from pyrrolnitrin, an antibacterial substance produced by Pseudomonas pyrrocinia. Although its mode of action has not been completely elucidated, fludioxonil has been shown to disrupt the osmoregulation pathway by activating Hog 1-type mitogen-activated protein kinase (Nakano 2020). Schnabel et al. (2021) investigated the mycelial growth of CGSC isolates, including four C. fructicola isolates, on media containing 1 mg/mL, 10 mg/mL, and 100 mg/mL fludioxonil. The CGSC isolates grew at a rate of approximately 30% with 1 mg/mL fludioxonil and retained the same growth rate up to 100 mg/mL fludioxonil. The ability to grow mycelia on fludioxonil-containing media suggested that CGSC, including C. fructicola, were intrinsically tolerant to fludioxonil. This tolerance might be facilitated by a mechanism unrelated to osmoregulation pathway disruption (Schnabel et al. 2021). As type II isolates grew at rates of approximately 20% at 100 mg/mL fludioxonil, it can be assumed that the C. fructicola field isolates collected in the present study also possessed the intrinsic tolerance proposed by Schnabel et al. (2021). In contrast, type I isolates grew at rates of > 50% at all fludioxonil concentrations < 100 mg/mL in the present study, In addition, 10 of the 14 type I isolates exceeded 30% in growth rate, even at 1000 mg/mL fludioxonil. Although the mechanism underlying the resistance of type I isolates to fludioxonil remains unclear, intrinsic tolerance alone cannot endow type I isolates with higher mycelial growth rates than CGSC isolates (Schnabel et al. 2021).

To the best of our knowledge, this is the first report of fludioxonil resistance in C. fructicola field isolates. Type I isolates were the minority in the strawberry anthracnose pathogen population in Saga Prefecture and were predominantly localized in Karatsu City. To date, no reduction in the effect of fludioxonil against strawberry anthracnose has been observed in general farm fields in the Saga Prefecture, including Karatsu City. However, the frequent sexual reproduction of C. fructicola might cause the rapid spread of fungicide resistance in the fungal population (Prihastuti et al. 2009). In addition, phenotypes of fludioxonil resistance associated with multidrug resistance have been found in other fungal species (Kuang et al. 2011; Zhou et al. 2020). Therefore, the dynamics of fludioxonil-resistant isolates in strawberry anthracnose populations must be monitored in Saga Prefecture. Moreover, the molecular mechanisms involved in the resistance of type I isolates warrant further elucidation. Therefore, future studies are required to facilitate the estimation of the risk of multidrug resistance caused by the spread of fludioxonil-resistant isolates in strawberry anthracnose populations.

Acknowledgements

We would like to thank Editage (www.editage.jp) for English language editing.

Author contributions

SA and JK designed the study and the main conceptual ideas. MK developed the statistical analysis plan and conducted sequencing and phylogenic analyses. NT contributed to the interpretation of the results. AF wrote the original manuscript with support from YI and MK. YI and MK supervised this study. All authors reviewed the manuscript draft and revised it critically on intellectual content. All authors approved the final version of the manuscript to be published.

Conflict of interest The authors declare that they have no conflict of interest.

Ethics approval Not applicable.

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Fig.S1Furutaetal.pptx
Fig. S1. Locality of the Colletotrichum field isolates collected in the present study.
Fig.S2Furutaetal.pptx
Fig. S2 The taxonomic status of Colletotrichum isolates from strawberry in Saga Prefecture, as inferred from phylogenetic analysis based on sequences of the intergenic region of apn2 and MAT1-2-1 (ApMat).
TableS1Furutaetal.xlsx
Table S1 Species identification of the Colletotrichum field isolates used in this study.
TableS2Furutaetal.xlsx
Table S2 The effect of fludioxonil on the mycelial growth of Colletotrichum fructicola isolates collected from strawberries in Saga Prefecture.

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First report of fludioxonil resistance isolate of Colletotrichum fructicola emerging on strawberry in Japan

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Abstract

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Introduction

Material and methods

Sample collection

Sensitivity assay for mycelial growth to fludioxonil

Inoculation test for evaluating the control efficacy of fludioxonil

Results

Sensitivity of mycelia to fludioxonil

Discussion

Declarations

References

Supplementary Files

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