Characterizations of Colletotrichum spp., Pathogens on Mango Fruits (Mangifera indica L. cv. ‘Nam Dok Mai’)

Sasivimol Laksanaphisut1/ Pattavipha Songkumarn1/ Somsiri Sangchote1/*

Received 27 Mar 2019/Revised 07 Jun 2019/Accepted 25 Jun 2019

ABSTRACT Mango anthracnose disease is caused by several species of Colletotrichum including C. gloeosporioides, C. acutatum, C. siamense and C. asianumand. The disease is considered as one of the major constraints of Thai mango export. However, the classification of these pathogens was still unclear. Generally, Colletotrichum spp. are classified based on their morphology. The aims of this study were to characterize and confirm the species of Colletotrichum isolates collected from mango orchards for exporting located in 6 different . The characterization was based on pathogenicity test, morphological and molecular characteristics. Forty four isolates were obtained from the symptomatic fruits. Pathogenicity test of all isolates showed typical anthracnose symptoms on the tested mango fruits and Koch’s postulates were fulfilled by re-isolation from the inoculated fruits. The morphological characterization identified all isolates as C. gloeosporioides with 6 morphotypes, as their conidia were hyaline, cylindrical with rounded-end and approximately 14.90x4.02 µm in size. Setae on infected tissue were also observed is some isolates. Species specific primer analysis could confirm 39 isolates of Colletotrichum as C. gloeosporioides. Ribosomal DNA (rDNA) gene sequencing and homology analysis of other five isolates revealed that these isolates were C. gloeosporioides except for the WH9 isolate which was identified as C. acutatum. By combining the results from molecular analysis with morphological characterization and pathogenicity test, we report that C. gloeosporioidesis is the main causal pathogen of mango anthracnose in the mango orchards for exporting in Thailand.

Key words: Colletotrichum gloeosporioide, C. acutatum, anthracnose, mango

1/ Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, 50 Ngam Wong Wan Rd, Lat Yao, Chatuchak, 10900, Thailand * Corresponding author: [email protected]

Thai Agricultural Research Journal Vol. 37 No. 2 May - August 2019 197 Introduction wounded and unwounded inoculations of

Thailand is among the world’s fif ive pathogenic Colletotrichum spp. led to the largest net exporters of fresh mango fruit disease development, different disease (FAOSTAT, 2012) with the cultivated area levels were observed as shown in the study of 1,967,904 rai giving the production of Prihastuti et al., (2009). The application approximately 1,462 kg/rai in 2017 (Offif ice of molecular techniques has been advocated of Agricultural Economics, 2017). The to characterize species of several plant cultivated areas of exported mango pathogenic fungi including Colletotrichum (cv.‘Nam Dok Mai’) in Thailand are spp. Random amplified polymorphic Phitsanulok, Nakhon Ratchasima, Sakeao, DNA (RAPD) marker had been used , Chiang Mai and Chchoengsao to separate C. gloeosporioides from provinces. One of the major constraints of C. fragariae, a causal agent of strawberry Thai mango export is anthracnose disease anthracnose (MacKenzieet al., 2008), and caused by Colletotrichum spp. Among them, Internal Transcribed Spacer (ITS) of C. gloeosporioides and C. acutatum were rRNA gene was widely applied to frequently reported to be the causal identify Colletotrichum spp. including agents of the disease (Rivera-Vargas et al., C. gloeosporioides, C. fragariae and 2006; Dhumtanom and Chantrasri, 2011). C. kahawae (Adaskaveg et al., 1997). Traditionally, Colletotrichum spp. have been In addition, ITS sequenceing analysis identified on the basis of morphological was able to separate C. acutatum, characteristics such as conidial size C. simmondsii and C. fragariae from and shape, appressorium shape and the other strawberry anthracnose pathogens pathogenicity test (Martínez-Culebras et al., (Anderson et al., 2012). Although several 2000); however, these methods are often species such as C. gloeosporioides, a main unreliable for species classification (Rive- fungus and C. acutatum, a minor one have ra-Vargas et al., 2006) due to the fact that been reported to be associated with mango morphological features expressing on the anthracnose disease in the study of culture media may differ from those Rivera-Vargas et al. (2006), the identifif ication expressing on the host plant. As shown in of these fungal pathogens is still ambiguous the study of Smith (1990), C. fragariae in Thailand. The present study aimed to associated with strawberry in the characterize and confif irm the main species southeastern United States produced setae of Colletotrichum associated with mango when they were observed on the plant fruit anthracnose obtained from different tissue but not the media culture. Although cultivated areas of Thailand by 1) determining

198 วารสารวิชาการเกษตร ปีที่ 37 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2562 their pathogenicity on unwounded fruits for further studies. 2) evaluating their morphological characteristics both on the host and 2. Pathogenicity testing culture media 3) Molecular analyzing Before being inoculated with the using species-specifif ic primers for tested isolates, mature green mango fruits C. gloeosporioides (CgInt/ITS4) and were dipped in hot water at 55°oC for 5 min C. acutatum (CaInt2/ITS4) and DNA followed by dipping in cold water for 5 min sequencing analysis of rDNA gene. and left untill completely dried. The unwounded fruit was artificially inoculated Materials and Method with 25 µ l of conidial suspension (3x106 1. Sample collection and isolation conidia/ml) of each individual Colletotrichum Ten mango fruits were obtained isolate and was subsequently covered from each mango exported orchard in with sterile Whatman No.1 filter paper disc Phitsanulok, Nakhon Ratchasima, Sakeao, (0.5x0.5 cm) maintain moisture of the Prachinburi, Chiang Mai and Chachoengsao inoculum. Each fruit was inoculated at 3 provinces, Thailand during April-July of positions including 2 cm from stem end, in 2013-2014. Locations were recorded. The the middle and 2 cm from stylar end on one samples were incubated at room side of the fruit. For control treatment, each temperature (25± 5°oC) until ripening. Fruits fruit was dropped with 25 µ l of sterile showing anthracnose symptoms were distilled water. All inoculated fruits were isolated for the causal pathogens by tissue maintained under high relative humidity by transplanting method. Advanced margins of covering with moist plastic bag at 25± 5°oC infected tissues were cut about 0.5x0.5 cm for 24 h. The plastic bag was then removed in size and surface disinfected with 1% and inoculted fruits were further incubated sodium hypochlorite solution for 3 min, at 25-28ºoC for 10 days, totally 4 inoculated then rinsed 2 times, with sterilized distilled fruits per each isolate were determined for water for transferred onto potato dextrose disease incidence (%). agar (PDA) plates and incubated for 7 days at 25± 2°oC under alternate white and near 3. Morphological characterization ultraviolet light 12 h light/dark cycle. Pure The morphological characteristics culture of each Colletotrichum isolate was were investigated on infected tissue and obtained by single conidia isolation and culture media. Conidial suspension of each was kept either on potato carrot agar (PCA) Colletotrichum isolate was prepared from at 15°oC or in 15% glycerol solution at -70°oC 7-day-old culture with distilled water at

Thai Agricultural Research Journal Vol. 37 No. 2 May - August 2019 199 3x106 conidia/ml then artificially inoculated 5-day-old culture was transferred and by spraying on unwounded mango fruit placed at the center of a 90 mm Pyrex@ surface and incubated at 25-28°oC for petri dish containing 30 ml Difco® PDA 10 days. Conidial, mass color, acervulus and media. The cultured plates were incubated setae characteristics were observed at 25± 2°oC under alternate white and near on fresh and thin section of the diseased ultraviolet light 12 h light/dark cycle with fi lesion which was mounted on the slide with f ive replications for each isolate. After lactoglycerol and investigated under compound incubation for 7 days, the radial growth of microscope at 40x magnification. Conidial mycelia (mm) was measured daily, and size was measured with Axio Vision Rel. 4.9.1 radial growth rate was then calculated. In software at 100 conidia per replication. addition, the colony color of each isolate Totally, 4 independent replications were was recorded. performed. Appressorium and colony characteristics were observed on PDA 4. Molecular characterization culture with mycelium newly isolated from 4.1 Genomic DNA extractions the disease symptom area. Appressorium Each isolate of Colletotrichum spp. was produced using a slide culture was cultured in 75 ml of MB [Malt broth: technique (Weir et al., 2012). Mycelium plug malt extract (Merck)] for 2-3 days at room of each Colletotrichum isolate was cultured temperature (25± 2ºoC) on orbital shaker at on PDA culture. Sterile cover slips (0.5x0.5 120 rpm. Young mycelia were harvested cm) were plugged into the culture media and transferred to 1.5 ml sterile eppendorf half way of its height and subsequently tubes. Mycelia were grounded in 200 µ l incubated at 25±+2°oC for 2 weeks. Vegetative solutionI (0.1 M NaCl, 0.2 M sucrose, 0.01 hyphae were grown on the surface of M EDTA and 0.03 M Tris HCl) containing the cover slip to produce appressoria. 10 µ l protease K, then added with 200 µ l The cover slip was removed and placed on solution II (50 mM Tris HCl, 50 mM EDTA a lactoglycerol droplet on the glass slide. and 2.5% SDS) and incubated at 65°oC Appressorial shape was characterized for 15 min. Subsequently, each tube was under compound microscope at 40x added with 128 µ l solution III (73.14 g magnification. Colony characteristics potassium acetate and 28.75 ml acetic acid including growth rate and colony color on in total volume at 300 ml of sterile water) the Difco® PDA media were observed. and 300 µ l of chloroform then mixed gently For each isolate, 5 mm diameter plug of and kept in ice for 10 min. The tube was mycelium from the colony margin of centrifuged at 14,000 rpm for 10 min. The

200 วารสารวิชาการเกษตร ปีที่ 37 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2562 supernatant was transferred to new 1.5 ml 50 µ l containing 1 µ l of 100 ng DNA, 5 µl sterile eppendorf tube then added 1 volume of 10X DreamTaq buffer, 2.5 mM each of of isopropanol into the supernatant, mixed dNTP at 4 µ l, 20 µ M of each primer at gently and centrifuged at 14,000 rpm for 2 µ l, 0.2 µ l of DreamTaq DNA polymerase 5 min. The supernatant was discarded (5 U/µ l) (Thermo Scientific; Watham, MA, and the pellet collected. Three hundred USA) and 37.8 µ l of distilled water. Reaction microliters of 70% ethanol was added was carried out in DNA Thermal Cycler in the tube to wash the DNA pellet and (MiniCyclerTM; Bio-Rad, Hercules, CA, USA) centrifuged at 14,000 rpm for 1 min. The with the following condition: 95°oC for 5 min, supernatant was discarded and 50 µ l of 30 cycles of 95°oC for 30 s, annealing of sterile water was added to dissolve DNA 62°oC for 30 s and 72°oC for 30 s, with a final in the tube. The DNA sample then was extension step of 72°oC for 5 min. The stored at -20°OC for further studies. species-specific primer, CaInt2/ITS4 was 4.2 Identification of Colletotrichum reported for C. acutatum identification species with CgInt/ITS4, CaIn2/ITS4 and (Brown et al., 1996). The species-specific ITS1/ITS4 primers primer of C. acutatum was amplified using Identification of Colletotrichum CaInt2 (GCCGCCGGCCCCGTCACGGGGG) isolates was performed by polymerase chain for forward primer and ITS4 for reverse reaction (PCR) with CgInt/ITS4, CaInt2/ITS4 primer. The negative control was without and ITS1/ITS4 primers. The species- DNA template and CaNR14 isolate of specific primer, CgInt/ITS4 was reported C. acutatum isolated from chili was used for C. gloeosporioides identification (Brown as positive control (CaNR 14 isolate was et al. 1996). The CgInt (GGACTCCCGCCTC- confirmed a species by DNA sequence CGGGCGG) for forward primer and ITS4 analysis of the 18S rRNA ITS region and it (TCCTCCGCTTATTGATATGC) for reverse shared 99% similarity with C. acutatum). primer were employed for PCR amplification. Reaction was performed in total volume The negative control was without DNA of 50 µ l containing 1 µ l of 100 ng DNA, template, and the MN001 isolate of 5 µ l of 10X Dream Taq buffer, 2.5 mM each C. gloeosporioides was used as positive of dNTP at 4 µ l, 10 µM each of primers at control (MN001 isolate was confirmed a 2 µl, 0.2 µ l of Dreams Taq DNA polymerase species by DNA sequence analysis of the (5 U/µ l) (Thermo Scientific, Watham, MA, 18S rRNA ITS region and it shared 99% USA) and 37.8 µ l of distilled water. Reaction similarity with C. gloeosporioides). PCR was carried out in a DNA Thermal Cycler reaction was performed in total volume of (MiniCyclerTM; Bio-Rad, Hercules, CA, USA)

Thai Agricultural Research Journal Vol. 37 No. 2 May - August 2019 201 with the following condition: 95°oC for 5 min, genomic DNA were subjected to amplify the 30 cycles of 95°oC for 45 s, annealing of ITS-fragments with ITS1/ITS4 primer set. 60.5°oC for 45 s and 72°oC for 45 s with a The DNA products were subsequently final extension step of 72°oC for 5 min. The purified and sequenced at Macrogen (Seoul, internal transcribed spacer (ITS) of rDNA Korea). DNA alignment was carried out was amplified using ITS1 (GCCGTAGGT- with Vector NTI Suite 9. The ITS-sequences GAACCTGCGG) for forward reaction and of unknown isolates were compared against ITS4 for reverse reaction (Martínez-Culebras the GenBank nucleotide database. et al. 2000). Reaction was performed in total volume of 50 µ l containing 1 µ l of 100 Results and Discussion ng DNA, 5 µ l of 10X Dream Taq buffer, 2.5 1. Sample collection and isolation mM each of dNTP at 4 µ l, 20 µ M each of Mango anthracnose in Thailand was primers at 2 µ l and 0.2 µ l of Dreams Taq reported to be caused by C. gloeosporioides DNA polymerase (5 U/µ l) (Thermo Scientific; group. However, identification of these Watham, MA, USA) and 37.8 µ l of distilled species particularly based on their water. Reaction was carried out in a morphology and disease symptoms cannot Biometra T Gradient Thermocycler clearly seperate them. In this study, we (Biometra GmbH, Göttingen, Germany) with characterized and confirmed the species the following condition: 94°oC for 5 min, of C. gloeosporioides group of mango 35 cycles of 94°oC for 1 min, annealing of anthracnose based on their pathogenicity 60.5°oC for 2 min and 72°oC for 2 min with test, morphological features mainly on the a final extension step of 72°oC for 10 min. host tissue and molecular characteristics PCR products were stained by novel juice, using PCR technique with two species- a non-mutagenic DNA staining reagent specific primers and ITS primers. Totally, (GeneDireX Inc., Taiwan) and were forty-four isolates of C. gloeosporioides group separated on a 1% w/v agarose gel. PCR were examined. Details of their origin were product sizes were determined using described in Table 1. HyperLadder™TM 1kb marker (BIOLINE, Lon- don, UK) with 1X TBE at 100V for 30 min. 2. Pathogenicity test PCR products were visualized under UV light Forty-four isolates of Colletotrichum and recorded with GelVue UV Transillumi- spp. obtained in this study were pathogenic nator (Syngene, Cambridge, UK). For the on the mango fruits under unwounded Colletotrichum isolates which were unable inoculation regime. The symptoms of to identify by species-specific primers, their anthracnose appeared as water soaked,

202 วารสารวิชาการเกษตร ปีที่ 37 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2562 Table 1 Isolates of C. gloeosporioides group from mango (Nam Dok Mai), geographical origins No. Isolates Geographic regions 1 PC1 Bang Khla, 2 PC5 Bang Khla, Chachoengsao province 3 PC7 Bang Khla, Chachoengsao province 4 PC12 Bang Khla, Chachoengsao province 5 PC23 Bang Khla, Chachoengsao province 6 PC28 Bang Khla, Chachoengsao province 7 PC40 Bang Khla, Chachoengsao province 8 PC51 Bang Khla, Chachoengsao province 9 Prachin1 Kabin Buri, Prachinburi province 10 Prachin5 Kabin Buri, Prachinburi province 11 Prachin7 Kabin Buri, Prachinburi province 12 Prachin8 Kabin Buri, Prachinburi province 13 Prachin10 Kabin Buri, Prachinburi province 14 Sakeao3 Mueang Sa Kaeo, Sa Kaeo Sakeao province 15 Sakeao6 Amphoe Mueang Sa Kaeo, Sa Kaeo Sakeao province 16 pn1.5 Pak Chong, Nakhon Ratchasima Province 17 pn8.3 Pak Chong, Nakhon Ratchasima Province 18 pn8.5 Pak Chong, Nakhon Ratchasima Province 19 pnf15 Bang Khla, Chachoengsao province 20 225 Pak Chong, Nakhon Ratchasima Province 21 227 Pak Chong, Nakhon Ratchasima Province 22 233 Pak Chong, Nakhon Ratchasima Province 23 237 Pak Chong, Nakhon Ratchasima Province 24 240/2 Pak Chong, Nakhon Ratchasima Province 25 240/3 Pak Chong, Nakhon Ratchasima Province 26 npf5 Noen Maprang, 27 npf6 Noen Maprang, Phitsanulok province 28 npf8 Noen Maprang, Phitsanulok province 29 npf10 Noen Maprang, Phitsanulok province 30 npf13 Noen Maprang, Phitsanulok province 31 np5 Noen Maprang, Phitsanulok province 32 wp16 Noen Maprang, Phitsanulok province 33 Prao7 Phrao, 34 Prao10 Phrao, Chiang Mai province 35 Thaton8 Tha Ton, Amphoe Mae Ai, Chiang Mai province 36 Thaton10 Tha Ton, Amphoe Mae Ai, Chiang Mai province 37 Thaton11 Tha Ton, Amphoe Mae Ai, Chiang Mai province 38 Thaton12 Tha Ton, Amphoe Mae Ai, Chiang Mai province 39 Thaton16 Tha Ton, Amphoe Mae Ai, Chiang Mai province 40 WH1 Wiang Haeng, Chiang Mai province 41 WH5 Wiang Haeng, Chiang Mai province 42 WH9 Wiang Haeng, Chiang Mai province 43 WH14 Wiang Haeng, Chiang Mai province 44 WH25 Wiang Haeng, Chiang Mai province

Thai Agricultural Research Journal Vol. 37 No. 2 May - August 2019 203 circular, black lesions with gelatinous Unlike the study of Liu et al., (2016), a orange, yellow-orange and yellow colour correlation between symptom types on conidial masses on the lesion (Figure 1 a, the pepper fruits in the field and some b). Pathogenicity of all tested isolates was Colletotrichum species such as C. truncatum confirmed on the mango fruits with and C. scovillei was observed but not in unwounded inoculation method. Despite other species. Therefore, based merely on Colletotrichum is known as the genus symptom characteristics it is still impractical containing serious pathogen species of to differentiate among Colletotrichum plants, they may also have an endophytic, species in the pepper fields. epiphytic and saprobic phase in their life cycle (Hyde et al., 2009). Furthermore, 3. Morphological characterization Colletotrichum spp. naturally infects on Traditionally morphological charac- the fruits directly, but pathogenicity tests in teristics have been widely used for fungal the past were mostly examined using the classification. In Colletotrichum, conidial and wounded inoculation method. This approach appressorial size and shape, appressorial may cause misinterpretation on their shape, the presence or absence of setae, pathogenic capability, since symptoms colony color, conidial mass color and growth appearing after inoculation may be caused rate have been used for this purpose. Based by the fungal isolate undergoing either on the morphological characteristic including saprophytic or weak parasitic phase instead conidial and appressorial size and shape, of pathogenic phase (Hyde et al., 2009). colony color and the presence or absence The unwounded inoculation technique that of setae were described by Sutton (1980). mimics the natural infection, could ensured Unlike this study, the usefulness of that all isolates investigated in this study morphotype grouping based on certain were the actual pathogens rather than characteristics for Colletotrichum endophytic Colletotrichum isolates acting identification was noted. In this study the as opportunistic pathogens. As no average conidial size of C. gloeosporioides differences of symptom characteristic could was 14.86± 0.09 (12.69-17.61) µ m in length be observed after inoculation with all isolates and 4.03 0.02 (3.60-5.17) µmin width with obtained in the study, it suggested that the length:width (L:W) ratio of 3.70± 0.3 the symptom characteristics of mango (3.20-4.52). Conidial shape was hyaline, anthracnose are not reliable criteria for one celled, cylindrical with rounded end. Colletotrichum species differentiation.

204 วารสารวิชาการเกษตร ปีที่ 37 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2562 appressorial size and shape, colony color and the presence or absence of setae were described by Sutton (1980). Unlike this study, the usefulness of morphotype grouping based on certain characteristics for Colletotrichum identification was noted. The average conidial size of C. gloeosporioideswas 14.86±0.09(12.69-17.61) µm in length and 4.03 ±0.02 (3.60-5.17) µmin appressorial size and shape, colony color and the presence or absence of setae were described width with the length:width (L:W) ratio of 3.70±0.3 (3.20-4.52). Conidial shape was hyaline, by Sutton (1980). Unlike this study, the usefulness of morphotype grouping based on certain one celled, cylindrical with rounded end. characteristics for Colletotrichum identification was noted. The average conidial size of C. gloeosporioideswas 14.86±0.09(12.69-17.61) µm in length and 4.03 ±0.02 (3.60-5.17) µmin widtha with the length:width (L:W)b ratio of 3.70±0.3 (3.20-4.52).c Conidial shape was hyaline, one celled, cylindrical with rounded end.

a b c

d e f

d e f

Figure 1 MorphologicalMorphological characteristics and pathogenicity testtest of of C.C. gloeosporioidesgloeosporioides PC7 PC7 Figure 1 isolate.isolate. aa)The The symptom symptom on on unwounded unwounded fruits.fruits. bb) ConidialConidial massmass onon thethe diseaseddiseased lesion.lesion. cAcervuli.c) Acervuli. d d) Appressoria. Appressoria. e e) Colony Colony appearances appearances on on PDA PDA media.media. f)f Conidia.ScaleConidia. Scale barbar 5050 µ m..

Figure While, 1 Morphological the average characteristics conidial size ofand pathogenicitywith rounded test end of C. and gloeosporioides irregular shape PC7 appressorium. Mycelium growth rate on isolate.C. acutatum aThe was symptom 13.91 (10.83-17.55) on unwounded µ m infruits. b Conidial mass on the diseased lesion. PDA medium was 3.4 mm/day and white clengthAcervuli.a and d3.85 Appressoria. (2.73-5.19) e µ Colonym in bwidth appearances with on PDA media.c f Conidia.Scale bar 50 µm. the length:width (L:W) ratio of 4.03. Conidial with orange masses. The setae were not shape was hyaline, one celled, cylindrical produced (Figure 2).

a b c

e d f

d e f

Figure 2 Morphological characteristics and pathogenicity of C. acutatum WH9 isolate. a) The symptom on unwounded fruits. b) Conidial mass on the diseased lesion. c) Acervuli. d) Appressoria. e) Colony appearances on PDA media. f) Conidia. Scale bar 50 µ m.

Thai Agricultural Research Journal Vol. 37 No. 2 May - August 2019 205 Furthermore, appressorial characteristics at the margin (Figure 3b) with 4.72± 0.05 were pale to dark brown, ovoid, clavate (3.40-5.60) mm/day of the mycelium and irregular (Figure 1f). Most isolates growth rate. The 3rd morphotype included were not able to produce setae on the ten isolates having olive and white color infected tissues except three isolates colony with no conidial mass. The average which were 233, npf8 and Prao7. With the of mycelium growth rate of this group was morphological characteristics described 4.78± 0.12 (3.77-5.83) mm/day (Figure 3c). above, all isolates obtained in this study The 4th morphotype contained three were identified as C. gloeosporioides, as isolates that were ochre in the origin with they were fitted into the current classification white at the margin and no conidial mass. of C. gloeosporioides described by Sutton The average of mycelium growth rate was (1980). Furthermore, based on colony colour 4.70± 0.02 (4.50-5.00) mm/day (Figure 3d). and conidial masscolour appearance on the The 5th morphotype had only one isolate infected tissues, all isolates were divided with gray to white colony and no appearance into six morphotypes (Table 2, Figure 3). of conidial mass. The average of mycelium The 1st morphotype comprised seventeen growth rate was 5.90 (3.10-9.60) mm/day isolates having white colour colony with (Figure 3e). The 6th morphotype included no appearance in their conidial mass. three isolates having gray to white near to The average of mycelium growth rate of this the margin of the colony with orange group was 5.03± 0.04(4.10-6.40) mm/day conidial mass. The average of mycelium (Figure 3a). The 2nd morphotype consisted growth rate was 4.30± 0.03 (3.80-5.20) of eleven isolates having a gray colony mm/day (Figure 3f). with orange conidial mass and white color

206 วารสารวิชาการเกษตร ปีที่ 37 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2562

1,

PC 8.3, 8.5,

np2,

WH5,

PC51, 8, pn

pn

10

Prachin

1.5,

PC28, np9, Thaton11, Prao

pn Mai ’ Prachin 8, 6, npf5,

npf10,WH1,

12,

13 15,

PC23, d f

233,

Dok

Isolate

npf 16 240/3, pnf

WH14,

sakeao

6, Thaton 225,

25,Thaton

7,

10, 3, 5, 10

npf ‘Nam 12,

Prachin7, PC40,

Thaton

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16

PC

Thaton Prachin sakeao 237, 7, wp PC5, npf8, WH PC1, Prachin 240/2, np5 fruits

a mango

Disease from

(66.67-100.00) (75.00-100.00) (83.33-100.00) (75.00-100.00) (100.00-100.00) (100.00-100.00) incidence (%) isolated

3.53 100 3.72 90.20 3.75 94.70 3.75 94.17 3.40 91.67 3.60 100 spp. 14.53 14.80 14.66 15.02 14.52 15.81 (11.07-19.55) (12.69-17.43) (12.71-16.27) (13.01-16.32) (13.62-15.09) (14.33-17.61)

Colletotrichum

of error.

4.12 3.99 3.92 4.02 4.27 4.41 color (2.93-5.48) (3.62-4.58) (3.71-4.28) (3.60-4.24) (4.08-4.45) (4.01-5.17)

standard

±

ith ith ith ith ith ith w w w w w w colony

end end end end end end

Conidia characteristics on mango fruit lesion mean on

Shape Width (µm) Length (µm) L:W ratio were

Cylindrical rounded rounded rounded rounded rounded rounded Cylindrical Cylindrical Cylindrical Cylindrical Cylindrical based

Data

0.04 0.05 0.03 0.02 0.03 5.90 5.03± 4.72± 4.75± 4.70± 4.30± (mm/day) Mycelium (3.10-9.60) (4.10-6.40) (3.40-5.60) (3.80-5.80) (4.50-5.00) (3.80-5.20) PDA medium inoculation. morphotypes growth rate on

after

gray distinct no with

white

colour, with

days

color, color

and six with

and orange 10

colony, mass mass colony at

mass

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with colony colony

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color,

origin

margin of

of mass

white the

the

mass mass

white white conidial conidial on PDA media conidial

at origin margin margin, colony at

and

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no no

measured conidial

the the the

characteristics of colony

White conidial White of orange Olive with Ochre at no Gray with Gray at conidial Summary were

Mor- e photype st nd rd th th th Lesions 4

6 2 5 3 1 Table 2 a

Thai Agricultural Research Journal Vol. 37 No. 2 May - August 2019 207

a b

c d

e f

Figure3Figure 3Six Six distinct distinct morphotypes morphotypes of C. of gloeosporioides C. gloeosporioides isolated isolated from mango from fruitsmango cv. fruits‘Nam st nd Dok‘Nam Mai’ Dok on Mai PDA’ on mediaPDA media after incubatedafter incubated at 25±2°C at 25± for2°O C7 fordays. 7 days.a 1 a) morphotype. 1st morphotype. b 2 rd th th th morphotype.b) 2nd morphotype. c 3 morphotype. c) 3rd morphotype. d 4 morphotype. d) 4th morphotype. e 5 morphotype. e) 5th morphotype. f 6 morphotype. f) 6th morphotype

ThanThan et et al .,al .,(2008) (2008) reported reported a correlationa medium. between Previous colony s tudycharacteristics reported ofthat C. gloeosporiodescorrelation between from chilicolony (Capsicum characteristics spp.) anthracnose C. gloeosporioides in Thailand wasand phylogeneticnot able to produce groups differentiatedof C. gloeosporiodes by using thefrom information chili (Capsicum on ITS andsatae β-tubulin on the regions. culture Likewise, media (DammNguyen et et alal.,., (2009)spp.) anthracnose and Prihastuti in Thailandet al., (2009) and phylogenetic showed the usefulness2012), however, of characters contradictory forC. gloeosporioides result was identificationgroups differentiated including by usingcolony the colour, information conidial shapes,observed conidial in this length study. and We width, found mycelial that growthon ITS rate, and pathogenicity-tubulin regions. test andLikewise, substrate -utilizationsome isolates on ammonium of C. gloeosporioides tartrate medium. can PreviousNguyen studyet al., reported(2009) and that Prihastuti C. gloeosporioides et al., wasproduce not able setae to produce in acervuli satae onof theinfected culture media(2009) (Damm showedet theal., usefulness2012); however, of different contradictory mango result tissue. was observedTherefore, in thesethis study. criteria We characteristic for C. gloeosporioides should be taken into account for found that some isolates of C. gloeosporioides can produce setae in acervuli of infected identification including colony color, identification of C. gloeosporioides. Some mango tissue. Therefore, these criteria should be taken into account for identification of C. conidial shapes, conidial length and width, studies reported the disadvantage of gloeosporioidesmycelial growth. Somerate, pathogenicity studies reported test theand disadvantage morphological of morphological characterization characterization as a mean as a smeanubstrate-utilization for Colletotrichum on ammonium identification tartrate (Freemanfor Colletotrichum et al., 1998). identification This approach (Freeman is still uncertain due to the fact that morphological features may express differently on the media and the host plant which may lead to the confusion on the identification of this species.

208 วารสารวิชาการเกษตร ปีที่ 37 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2562 et al., 1998). This approach is still uncertain showed 98% sequence similarity with due to the fact that morphological features C. acutatumde posited in the Genbank may express differently on the media and (Table 4). Therefore, the application of the host plant which may lead to the molecular techniques has been advocated confusion on the identification at species to characterize species of several plant level. pathogenic fungi including Colletotrichum spp. In our study, species of C. gloeosporioides 4. Molecular characteristics group on mango fruits can be resolved Species specific primers for into two species: C. gloeosporioides and C. gloeosporioides (CgInt/ITS4) and for C. acutatum, by combining PCR based C. acutatum (CaInt2/ITS4) were used identification with species-specific primers to confirm the species of fourty-four and DNA sequencing of ITS region. In fact, Colletotrichum isolates. Thirty-nine out we found that there may be some nucleotide of 44 isolates were identified as variations on Internal Transcribed Spacer C. gloeosporioides, since 450 bp fragments (ITS) of rRNA gene, since the failures of were amplified with the C. gloeosporioides PCR amplification with those primers specific primers. No amplified PCR in some isolates did exist. Higher product was obtained from seven isolates resolution of the genetic relationships of including PC51, pn1.5, npf13, Prao7, WH1, species within a genus can be achieved by WH9 and WH25 (Table 3), when the same using gene multilocus analysis (Martens species-specific primers were used. In the et al., 2008). Lima et al., (2013) classified case of PCR amplification with C. acutatum the causal agent of mango anthracnose specific primers, none of isolates produced in northeastern Brazil by nucleotide the PCR product except the positive control, sequencing of these following locus: C. acutatum isolate CaNR14 (Table 3). glyceraldehyde-3-phosphate dehydrogenase, Sequencing and homology analysis of rDNA actin, -tubulin, calmodulin, glutamine gene using ITS1/ITS4 primers in the seven synthetase and rDNA-ITS. Therefore, further unidentified isolates revealed that the PC51, analysis on such multilocus of all isolates of pn1.5, npf13, Prao7, WH1 and WH25 isolates C. gloeosporioides group obtained from showed 97-99% sequence similarity with mango in this study should be carried out C. gloeosporioides, whereas the WH9 isolate in the future.

Thai Agricultural Research Journal Vol. 37 No. 2 May - August 2019 209

positive primers; pecific

as

(ITS)

used species-s species

was

ITS1/ITS4

C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides Unidentified C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides Unidentified C. gloeosporioides Unidentified C. gloeosporioides Unidentified using

and

MN001 by

a (Ca) + + + + + + + + + + + + + + + + + + + + + + + + + +

template.

------anthracnose DNA CaInt2/ITS4

-- + + + + + + + + + + + + + + + + + + + + + +

Cg Ca ITS Primer reaction Mai’ (Cg),

without

(ITS) Dok

was

. CgInt/ITS4

‘Nam primer

control with

fruit Prachin7 Prachin8 Sakeao3 Prachin10 Sakeao6 pn1.5 pn8.3 pn8.5 pnf15 225 233 237 240/2 240/3 npf5 npf6 npf8 npf10 np2 Thaton12 Thaton16 WH1 - - WH5 WH9 - - WH14 WH25 - -

C. acutatum

of

ITS1/ITS4 negative mplified

mango a for

The and

were from

control (Ca)

DNA

species Isolate determined. isolates

positive

not as C. gloeosporioides C. acutatum C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides Unidentified C. gloeosporioides

C. gloeosporioides Unidentified C. gloeosporioides C. gloeosporioides C. gloeosporioides Unidentified C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides

genomics spp. =

CaInt2/ITS4

nd The used

a

a + + + + + + + + + + + + + + + + + + + + nd was

primer twice

absent.

------+ least nd nd

Colletotrichum CaNR14

at f

product o

- nd nd ------+ + + + + + + + + + + + + + + + + nd - nd nd nd - Cg Ca ITS Primer reaction PCR whereas

repeated =

species-specific -

were Cg Ca ITS

Cg Ca nd ITS nd nd

(Cg), of of of identification

of of of

present,

Isolate control control control C. gloeosporioides

control control control

Species product

a CgInt/ITS4

for

PCR

PCR

=

Negative Positive Negative Positive Negative Positive PC1 PC5 PC7 PC12 PC23 PC28 PC40 PC51 Prachin1 Prachin5 npf13 np5 np9 wp16 Prao7 Prao10 Thaton8 Thaton10 Thaton11 primer Table 3 All mplifications + control

210 วารสารวิชาการเกษตร ปีที่ 37 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2562 sequences

GenBank

C. gloeosporioides

some

and

spp.

97% AJ311883.1 C. gloeosporioides 98% AF272782.1 C. acutatum 99% KF053199.1 C. gloeosporioides 99% KT582187.1 C. gloeosporioides 98% KC816034.1 C. gloeosporioides 99% KF177685.1 C. gloeosporioides 97% KM111484.1 Colletotrichum

of

isolates

seven

province between

province

province province province province

identity

Mai Mai Mai

Ratchasima

province

Phitsanulok

Mai Chiang Chiang Chiang sequence

Nakhon Chachoengsao

ITS

of Chiang Haeng , Haeng , Haeng ,

Maprang, Khla,

Chong,

result

Wiang Wiang Wiang BLAST

WH25 WH9 WH1 Prao7 Phrao, npf13 Noen pn1.5 Pak PC51 Bang Isolates Geographic location in Thailand % identity GenBank accession no. Table 4

Thai Agricultural Research Journal Vol. 37 No. 2 May - August 2019 211 Conclusions C. gloeosporioides group. Therefore, more This study provides the evidence to isolates of the anthracnose pathogen from confirm the identity of causal agents of high number of sample fruits and various anthracnose disease on mango fruits sampling locations are needed for further in Thailand. By reclassification of investigation. Furthermore, molecular C. gloeosporioides group with pathogenicity characterization by analyzing sequencing test, morphological and molecular data from other locus rather than ITS of characterizations, C. gloeosporioides is rRNA gene is suggested for in-depth reported to be the main cause of fruit species confirmation. anthracnose while C. acutatum is minor cause. A number of techniques facilitating Acknowledgement reclassification of C. gloeosporioides group I gratefully acknowledge the were reaffirmed here in this study and will kind assistance of Department of Plant be useful for plant pathologists to confirm Pathology, Faculty of Agriculture, Kasetsart these species identity. For examples, wounded- University, Bang Khen campus, Thailand for inoculation method on mango fruit may not my Ph.D. work possible. be required for pathogenicity test since high disease incidences can still be obtained with References unwounded inoculation. We also suggested Adaskaveg, J.E. and R.J. Hartin. 1997. that only some morphological characteristics, Characterization of Colletotrichum i.e., conidial and appressorial shape and acutatum Isolates Causing mycelium growth rate could be adopted for Anthracnose of Almond and differentiating between C. gloeosporioides Peach in California. Phytopathol. and C. acutatum. Furthermore, molecular 87: 979-987. technique by using PCR amplification with Anderson, J.M.; E.A.B. Aitken, E.K. Dann and species-specific primers, i.e., CgInt/ITS4 for L.M. Coates. 2012. Morphological C. gloeosporioides and CaInt2/ITS4 for and Molecular Diversity of C. acutatum and DNA sequencing of Colletotrichum spp. Causing Pepper Internal Transcribed Spacer (ITS) of rRNA Spot and Anthracnose of Lychee gene are useful for identifying the species (Litchi chinensis) in Australia. Plant of C. gloeosporioides group. As two species Pathol. 62: 279-288. of Colletotrichum; C. gloeosporioides Brown, A.E.; S. Sreenivasaprasad and and C. acutatum, are reported here, we L.W. Timmer. 1996. Molecular cannot rule out other species within Characterization of Slow-growing

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