Colletotrichum Species Related To “Nam Dork Mai See Tong” Mango In Central Thailand And Risk Detection of Carbendazim-Resistant Strains


 Nam Dork Mai See Tong mango is a deluxe commercial fruit in Thailand, however anthracnose disease becoming a major problem affecting market-driven. Fungicide treatment is the main component in the mango anthracnose management, notwithstanding the appearance of fungal-resistance has been become a factor of fungicide limition and can affect to increasingly higher costs. Thirty-two isolates of the Colletotrichum species complex were obtained from anthracnose-diseased mango cv. Nam Dork Mai See Tong in 3 provinces of Thailand. Fungal identification based on morphological and molecular markers was investigated. All isolates were divided into 3 species: C. asianum , C. gloeosporioides and C. siamense . Pathogenicity tests revealed that all 3 species caused symptoms on artificially unwounded mango fruits and leaves. Only C. gloeosporioides have previously been reported on mango, while C. asianum and C. siamense were first reports associated with mango in Thailand. The responsiveness of Colletotrichum isolates to carbendazim was evaluated using an MIC assay. Nine isolates of C. asianum were highly resistant, while 5 and 8 isolates of C. gloeosporioides were resistant (R) and highly resistant (HR), respectively. Moreover, all isolates of C. siamense were HR. Mutations were detected by PCR of a partial sequence of the β-tubulin gene. The sequence of β-tubulin gene in HR strains showed a single nucleotide transversion of adenine to cytosine, resulting in a substitution at codon 198. However, R strains were found to have only an amino acid change at codon 200. Additionally, PCR-RFLP was applied as a rapid technique to detect carbendazim resistance. The results demonstrated that only the Bsh 1236I restriction enzyme generated two bands (200 and 300 bp) in the highly resistant strain, but these bands were absent in the sensitive (S) and R strains. Hence, PCR-RFLP technique showed the potential to specifically detect benzimidazole fungicide resistance in 3 species of Colletotrichum. Moreover, this technique is accessible and feasible for laboratory assessment


79
In 2018-2019, world mango production expanded to more than 100 countries, with more than 44,626,000 tons of 80 fruits produced annually. Thailand is one of the top three mango-producing areas, producing almost 7.69% of 81 global mango production, and approximately 2% of fresh mango fruit is exported (Worldatlas 2019). Anthracnose

96
The first report of anthracnose disease management in mango was by Jeffries et al. (1990), in which fungicide 97 application was suggested for the management of this disease due to its ease of use and success in disease control 98 (Cools et al. 2005). Carbendazim, a member of the benzimidazole class, is a fungicide that is widely used for 99 anthracnose disease control and can be managed both in the field and postharvest. The mode of action of 100 carbendazim is the inhibition of mitosis and cell division (β-tubulin assembly in mitosis), but recently, the 101 appearance of fungal resistance has become important and has affected the ability to use the fungicide for 102 anthracnose management in the mango industry (Brent 1995). The report of the Fungicide Resistance Action 103 Committee (FRAC) (2020) explained that fungicides have become an integral part of efficient food production 104 and that the loss of a fungicide through resistance is a problem that affects everyone.

105
The methods for fungicide resistance detection require isolation of pathogens treated with each fungicide (Anon 106 1991), but these processes are time-consuming. Currently, molecular biology is a new method for fungicide 107 detection once the other mechanisms have been exhausted. For carbendazim, Ma and Michailides (2005) reviewed 108 the molecular mechanisms of fungicide resistance in four fungicide classes, including the benzimidazole class.

109
The results indicated that resistance was correlated with point mutations in the β-tubulin gene, resulting in altered

128
Hence, the rapid detection method was applied as an essential tool for fungicide resistance inspection to improve 129 fungicide utilization for anthracnose control. The aim of this research was to detect carbendazim resistance in

142
Petri dished containing 15 mL of PDA were inoculated with a 5 mm in diameter core taken from the edge of an 143 activity growing 5-day old culture. The culture was incubated at 25°C under a photoperiod of 12 hr light/12 hr 144 dark. Colony type and spore mass color were recorded at day 5. Fifty conidia were randomly selected from each 145 replicate to measure their length and width under compound microscope with Olympus CellSens Standard 146 software version 1.6. Appressoria were induced with modified slide culture technique (Johnston and Jones, 1997).

147
The isolates 5-day old cultures on PDA were transferred onto 25.476.2 mm sterile microscope slides and 148 covered with 2222 mm cover slips incubated in petri dish to serve as a moisture chamber under 25°C for 4 days.

159
PCR thermal cycling (Sensoquest GmbH, Göttingen, Germany) was carried out using the following program: 160 predenaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 30s, annealing for 30s (the temperature 161 each of primer is shown in Table 1) and extension at 72°C for 1 min, with a final extension step of 72°C for 10 162 min. PCR products were stained with GelStar® and verified on 1.2% agarose gels via electrophoresis in 1X TBE

227
During disease survey, anthracnose symptoms in the mango orchards were observed (Table S1). On mango, 228 disease symptoms of anthracnose were separated into 3 types: 1) 6.25% of the symptom on inflorescences are 229 dark-brown spots to elongated dark lesions which can enlarge, coalesce, and blighted flowers affecting small 230 emerging fruits that could fall out, then ( Fig. 1a-1b), 2) 21.87% of lesions on leaves start as brown to black small 231 spot, then developing to irregularly shaped lesions with brown-gray to black necrotic spots on leaf surfaces ( Fig.   232 1c-1d), and 3) 71.88% small black circular spots becoming sunken, rounded brown to black lesions on ripe fruits 233 and producing acervuli on the lesions with bright salmon, salmon-orange to pale colored spore masses at high  gloeosporioides species complex due to their cottony aerial mycelia and white to gray mycelia covered with pale 238 to orange spore masses ( Fig. 2a-2j). Conidia were straight to cylindrical with round ends ((3.59)6.91  239 11.02(19.52) µm) ( Fig. 2k-2t), no setae, and appressoria pale to dark brown with various shapes such as clavate, 240 long clavate to irregular ((4.37)7.31  6.49(11.59) µm) (Table S2; Fig. 2u-2ad; Fig. S1). Then, identification of 241 species complexes using PCR techniques based on multilocus phylogenetic analyses was performed.   Table 3. Ten isolates were absolutely 255 identified as C. asianum with 95% bootstrap values. Furthermore, three representative isolates were determined 256 to be C. gloeosporioides with approximately bootstrap value 86%, while two representative isolates (RB003, 257 RB006 and PC002) were accurately classified as C. siamense with high bootstrap values 96% shown in the branch 258 of phylogenetic tree (Fig. 3). All the sequences (excepted results of specific-primer) generated in this study were 259 deposited in GenBank and DDBJ for assigning accession numbers (Table 3).

261
Colletotrichum isolates RB005, CS008 and RB003 were selected from C. asianum, C. gloeosporioides and C. 262 siamense, respectively, in order to test their pathogenicity. Symptom on unwounded fruits and leaves developed 263 sunken spot, dark brown to brown lesion at day 5 after inoculation (Fig. 4). The damage of Colletotrichum 264 gloeosporioides had the highest infection incidence reached to 100% in both inoculated fruits and leaves, while 265 the C. asianum and C. siamense were about 75 and 60% on fruits and each absolutely counting 100% on leaves 266 (Table 4).

267
Carbendazim resistibility assays 268 Thirty-two isolates of Colletotrichum separated into 3 species were tested for carbendazim responsiveness by MIC 269 assay on PDA amended with carbendazim at concentrations of 0.1 -1,000 mg/L, including the recommended rate.

270
The results indicated that 9 isolates (90.00%) of C. asianum were found to be HR strains, while one isolate 271 (10.00%) was found to be an R strain. Six isolates (31.58%) of C. gloeosporioides were clustered as sensitive 272 strains, and 5 isolates (26.32%) were proven to be R strains; moreover, 8 isolates (42.11%) were revealed to be 273 HR strains. Ultimately, all 3 isolates (100.00%) of C. siamense were evaluated to be HR strains (Table 5).  (Fig. 5a 5b, 5f). Other point mutation within the remaining 283 codons, such as codons 167 and 240, were studied with the same pair of primers, and no differentiation was 284 detected. The results indicated that the mutated codons E198A and F200Y of the β-tubulin gene were related to 285 the responsiveness phenotype of the 3 Colletotrichum species causing mango anthracnose and were associated 286 with resistance to MBC fungicides, such as carbendazim, in this study.

288
The partial β-tubulin gene sequence amplified with the TB2L/TB2R primers was subjected to in silico RFLP using 289 pDRAW32 software to assess HR, R and S strains. The results showed that 37 of 86 restriction enzymes in the 290 program did not cut the PCR fragment. However, the BstUI restriction enzyme revealed distinctly different 291 fragments among the 3 strains (Fig. 6c, 6d). Digestion of the HR strain with Bsh1236I (isoschizomers: AccII,

292
BspFNI, BstFNI, BstUI, MvnI) generated bands of approximately 200 and 300 bp, while the S strain and R strain 293 products were not cut (Fig. 6b, 6e). The Bsh1236I enzyme recognizes the CG^CG site and cuts at the position of 294 codon 198, which was useful for distinguishing between HR and S strains. For the R strain, the HpyAV restriction 295 enzyme (isoschizomers: Hin4II) cut and showed differentiation between R and HR or S strains, but the cut and 296 recognition sites were not positioned at codon 200 (Fig. 6). Hence, RFLP using the Bsh1236I restriction enzyme

354
were sensitive to carbendazim. The results as mentioned in Table 5

362
Usually, fungicide resistance studies require isolation of the pathogen in a pure culture and subsequent plating 363 on a medium containing fungicide or a "poisoned food" (Anon 1982, 1991). The conventional method is time-364 consuming, which is why molecular techniques provide a new opportunity for rapid detection. Although molecular 365 tools are rapid techniques, most studies differentiate between S and R strains by using nucleotide sequencing 366 analysis as showed in Fig. 5a and 5b.

394
This research is not only identification but also differentiation among sensitive, resistant and highly resistant 395 strains in population of Colletotrichum species obtained from mango anthracnose disease by using virtual PCR-

396
RFLP and confirm with PCR-RFLP in gel electrophoresis. The method can be performed in a very short time and 11 achieved reliable data comparing to the old assay such as poisoned food or ED50 calculation, then PCR-RFLP 398 technique illustrated the potential to specifically detect benzimidazole fungicide resistance in 3 species of 399 Colletotrichum like C. asianum, C. gloeosporioides and C. siamense. Furthermore, this technique is accessible 400 and feasible for laboratory assessment. It is a crucial caution and signal for precise management using fungicide 401 utilization with the target to achieve effective control. Once, the mutation fungal strains invade in a field, it is 402 quite tough to reduce the population of these strains. Hence, a rotation of different fungicide groups is needed.

403
Moreover, this is the first report of carbendazim resistance of three species of Colletotrichum, causal agents of 404 mango anthracnose in Thailand using PCR-RFLP for benzimidazole fungicide resistant detection.

407
We would also like to thank the Mycology Laboratory

Conflicts of interest/Competing interests:
The authors declare no conflict of interest.

415
Code availability: Not applicable.           The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches at black arrows, and the evolutionary distances were computed using the aeschynomene), (b) Partial protein sequences of 9 isolates aligned with the reference sequence U14138 (colored according to the protein setting in MEGA version X), (c) PCR products subjected to 1.2% agarose gel electrophoresis, (d) Simulated digested products produced via in silico restriction digestion using by pDRAW3.2 with Bsh1236I, (e) Bsh1236I-digested product that distinguished between S and R strains, and (f) Secondary structure of β-tubulin; the arrows indicate codons 198 and 200.