Indian Phytopathology https://doi.org/10.1007/s42360-021-00454-9 RESEARCH ARTICLE Importance, etiology and management of the tear stain disease symptoms of mango fruits in Ghana Joseph Okani Honger1  · Stephen Narh1 · John B. Lambon2 Received: 23 March 2021 / Revised: 5 October 2021 / Accepted: 13 December 2021 © Indian Phytopathological Society 2022 Abstract The tear stain symptom refers to slightly raised blisters which reduce the aesthetic value of mango fruits in Ghana. The dis- ease incidence, severity and percentage of exportable mango fruits on infected mango trees were determined in the coastal savannah zone of Ghana in 2019. Samples of disease fruits were collected and the causative agent isolated and identified using phenotypic and genotypic characteristics. Selected copper based fungicides, namely, Cuprous oxide (750 g/kg), Copper oxychloride (500 g/kg) and Mancozeb (800 g/kg) either solely or in combination with Acibenzolar-S-methyl were applied in the field to determine their effect on the disease incidence and severity and on the percentage of exportable fruits. The disease incidence ranged from 9.4% to 11.0% in the major season and 37.3% to 45.0% in the minor season. Severity ranged from 0.08 to 0.1 in the major season and 1.4 to 2.1 in the minor season. The fungus isolated showed the characteristic short conical spores with rounded edges, an indication that it was C. gloeosporioides. The combination of gene sequences of the ITS region and the partial actin gene identified the fungus as C. siamense. All fungicides evaluated were able to reduce the disease incidence and severity on treated trees and resulted in a higher percentage of exportable fruits compared to non- treatment control trees. Keywords Mango · Anthracnose · Tear stain · Colletotrichum siamense Introduction GDP in the near future. It has been estimated that about 30% of Ghana’s total mango production is lost yearly to poor Mango (Mangifera indica) is one of the most important non- postharvest practices (MOAP 2016). Other factors, such as traditional exports fruit crops from Ghana (Ablormeti et al. pests and diseases infestation, contribute to reduced yields 2021). Total production of mango has been estimated to be in the field. Therefore, when these problems are addressed, about 110,000 tonnes. Out of this, about 40,000 tonnes are volumes of production and exports are likely to rise. consumed locally, making the crop an important food secu- In Ghana, one common disease of mango is tear stain. rity crop for the country. In terms of the Ghana’s total GDP, The symptoms of the disease are characterised by the aggre- mango exports contribute a modest 0.3%, however, with the gation of numerous tiny spots that are slightly raised and ever increasing demand for the crop in international markets, are rough to touch. The pattern created by the symptom on the crop has a potential of contributing more to the country’s the fruit surface sometimes resemble the back covering of an alligator, causing the symptoms to be described as an Joseph Okani Honger alligator skin effect (Nelson 2008). The nature of the blem-* johonger@yahoo.com ishes reduce the aesthetic value of the fruits and hence their Stephen Narh marketing, especially in international markets. sknarh@gmail.com The tear stain disease symptom is very common on John B. Lambon mango fruits produced in the minor season in the Coastal jlambon2000@yahoo.com savannah areas of Ghana, where huge quantities of the crop are produced for both the local and export markets. A cas- 1 Soil and Irrigation Research Centre, University of Ghana, ual look and reports by farmers show that several fruits, Legon, P.O. Box LG44, Accra, Ghana intended for international markets are rejected due to the 2 Council for Scientific and Industrial Research, Head Office, blemishes caused by the disease. Work, however, has not P. O. Box M32, Accra, Ghana Vol.:(012 3456789) Indian Phytopathology been carried out to determine the importance of the disease after which the district extension agents were consulted for in these major mango producing areas of Ghana. the selection of localities where the orchards were located. The etiology of the tear stain disease of mango in Ghana Mango farms (2–100 acres) in each locality were selected is uncertain. It has been attributed to expansion and contrac- at random and assessed for the disease incidence and sever- tion of fruits, in response to temperature changes. Others are ity. Diseased mango fruits were collected at random from of the view that it represents healing scars from wounds cre- farms selected at Akuse, Somanya and Kpong for isolation ated by insects feeding activities. Elsewhere, the disease has of causal agent of the disease. been ascribed to Colletotrichum gloeosporioides (Nelson, 2008), another possibility of the etiology of the disease in Determination of disease incidence and severity the country. Different species of Colletotrichum have been identified on mango in Ghana and have all been shown to Mango fruits, totalling 400 were randomly selected from produce the characteristic, dark brown slightly sunken spots, at least 20 trees per farm, were inspected for the presence known as anthracnose on mango fruits in Ghana (Honger of the disease symptoms (Fig. 1a and b) and a farm was et al. 2014). Currently, none of the species have so far been marked as free from the disease (0) or infected (1). A sub- associated with the tear stain symptoms. sample of 50 fruits per farm was taken and the surface area Copper based fungicides such as Funguran (copper of the fruit covered by the disease symptom was visually hydroxide) and copper oxychloride are one of the major rated on a scale of 0–5, 0 = no infection, 1 = up to 5% of group of fungicides, recommended for the control of dis- fruit surface area covered, 2 = 6–10% of fruit area affected, eases affecting mango in Ghana (EPA 2015). Copper-based 3 = between 11 and 20% of fruit area covered, 4 = 21–50% of fungicides have been shown to be as effective as other syn- fruit area affected and 5 more than 50% of the fruit surface thetic fungicides (Honger 2013). One advantage in the use area covered (Lakshmi et al. 2011). Data obtained were used of copper is that it is accepted in organic production systems to calculate the disease prevalence, incidence and severity worldwide, and can therefore be used for the control of dis- as follows: eases on fruits that are intended to be sent to international markets. Several of these fungicides are available on the No of farms with disease symptomsDisease prevalence = × 100 Ghanaian markets, but have not been evaluated extensively, Total numberof farms surveyed to determine their effect on the disease incidence and sever- ity of the tear stain disease symptoms on mango, in the No of infected fruitsDisease incident = × 100 country. Number of fruits inspected Given the paucity in information as regards the tear stain disease symptoms in Ghana, this work was carried out to ∑ fX determine the determine the disease incidence and severity Disease severity = ∑ where,x and its effect on mango disease marketing, to identify the causal agent of the disease and to evaluate some selected copper-based fungicides for the control of the disease in the f = number of fruits with a particular rating, x = a particu- field. lar rate based on the percentage of fruit surface area covered by disease lesion. Data collected from farms within the same locality were bulked and means and standard errors calcu- Materials and methods lated for the different localities. Field survey and collection of diseased mango Isolation and morphological characterisation fruits. of Colletotrichum species Field survey for the prevalence, disease incidence and Isolation of the causal agent was carried out in the Plant severity was carried out in commercial mango orchards the Pathology Laboratory of the Department of Crop Science, major (May–June) and Minor (December) mango produc- University of Ghana. The causal agent was first isolated on tions seasons of 2019. The surveys were carried out in five water agar (20 g/l) and sub-cultured on potato dextrose agar different locations in Ghana where mango farms are heav- (39 g/l). Each medium was autoclaved at 121 °C and after ily concentrated. These were Asutuare and Dodowa in the cooling were dispensed into clean sterilized Petri dishes. Greater Accra Region, Akuse, Somanya and Kpong in the Diseased fruits from the field were first washed with soap Eastern Region and Juapong in the Volta Region of Ghana. and rinsed under running water. Piece of the fruit tissues The districts where these localities were found were selected (5 × 3 mm) were then excised from the blisters on some of with the help of National Agricultural Extension agents the fruits, surface sterilised with 1% sodium hypochlorite, 1 3 Indian Phytopathology Fig. 1 Symptoms and cultural and morphological characteris- tics of the causal agent of tear stain disease of the tear stain disease of mango. a typical symptoms on a fruit with slight blisters in the field; b fruits showing the pronounced blister- like symptoms in the field, c symptoms induced on fruits artificially inoculated with the pathogen, d mycelial growth on PDA, e acervuli showing the presence of setae, f short coni- cal spores with rounded edges blotted dry with sterile paper tissues and plated on water Pathogenicity test agar. Other cleaned diseased fruits were stored in cardboard for two weeks at 25–27 °C and 65% RH, till the blister-like Pathogenicity of isolated Colletotrichum species was tested spots expanded to produce dark-brown spots on the fruits on physiologically matured mango fruits. Spore suspension surface. Pieces of the fruit tissues (5 × 3 mm) were then of the fungi (1 × 107 spores/ml) was used to inoculate, dis- taken from the advancing edge of the disease symptoms and infected healthy mango fruits, by placing the 15 μl of the plated also on water agar. At 7 days, when enough growth suspension on a filter paper disc attached to the fruit sur- of a fungus that grew was obtained, it was sub-cultured face. Sterile distilled water served as control. The inoculated on PDA and incubated for a further 7 days. Isolates that fruits were placed in plastic containers on benches in the grew were identified to the genus level based on cultural laboratory at 25–27 °C and 65% RH. When symptoms were and morphological features. Colletotrichum species which observed, the pathogen was re-isolated from the induced were consistently isolated from the expanded brown lesions symptoms to complete Koch’s postulates. and from very few of the blister-like spots were maintained. Single spore cultures of the selected isolates were produced Molecular characterisation and plugs (8 mm in diameter) were placed on fresh PDA plate and incubated at 28 °C. Diameter of colony growth was Polymerase chain reaction and sequencing of products measured daily for 7 days and the seven-day average of mean daily growth was calculated to represent the mycelial growth Five of the isolates of the Colletotrichum species were rate (millimetres per day). Spore sizes were measured under selected at random and DNA was extracted using the the microscope and means for 20 individual conidia per iso- Sigma’s GenFlute Plant Genomic DNA Miniprep Kit (St. late was calculated (Prihastuti et al. 2009). The slide cul- Louis, MO, USA), following the manufacturer’s instruc- ture technique after Johnston and Jones (1997) was used to tions. DNA extracted was used as templates in PCR. The stimulate appressorial growth after which the dimensions of PCR were carried out using two primers pairs; ITS1/ITS4, the appressoria produced were also measured. Lasiodiplodia to amplify the entire internal transcribed spacer region and Aspegillus species were isolated from few of the sam- (White et al. 1990) and ACT512F/ACT783R to amplify ples of the blister-like symptoms. Lasiodiplodia is known to a part of the actin gene (Carbone and Kohn 1999). PCR cause soft rot of mangoes in Ghana while Aspergillus spe- reaction mixtures and conditions were after Honger et al. cies were considered saprophytes on mango fruits in Ghana. (2014). Amplification products were separated by 1.5% These two fungal species were therefore not selected for any w/v agarose gel (Invitrogen, Carlsbad, CA), stained further studies in this work. with gel red. Separation was achieved with a 100 V and 1 3 Indian Phytopathology was run for 1.5 h. The presence and size of bands were Phylogenetic analysis observed under UV light. The amplified products were purified and sequenced directly at ETON Bioscience Lab- The concatenated nucleotide sequences of the ITS region oratory at Raleigh in North Carolina. Nucleotides were and the actin gene of 31 ex-types and isolates of Colletotri- analysed and consensus strands generated with the aid of chum, made up of 26 isolates of confirmed identities (down- Bioedit software. loaded from EMBL database) and 5 obtained from this study (Table 1), were aligned using Clustal W. Maximum Par- simony analysis was performed on the multiple sequence Table 1 Isolates of Colletotrichum used in the study with their accession numbers Species Strain identification Host Country GenBank accession num- bers ITS ACT C. aenigma ICMP 18608* Persea americana Israel JX010244 JX009443 C. aeschynomenes ICMP 17673*, ATCC 201874 Aeschynomene virginica USA JX010176 JX009483 C. alatae CBS 304.76*, ICMP 17919 Dioscorea alata India JX010190 JX009471 C. alienum ICMP 12071* Malus domestica New Zealand JX010251 JX009572 C. aotearoa ICMP 18537* Coprosma sp. New Zealand JX010205 JX009564 C. asianum ICMP 18580*, CBS 130418 Coffea arabica Thailand FJ972612 JX009584 C. asianum ICMP 18696 Coffea arabica Thailand JX 009,576 C. clidemiae ICMP 18658* Clidemia hirta USA JX010274 JX009537 C. cordylinicola MFLUCC 090551* Cordyline fruticosa Thailand JX010226 HM470235 C. fructicola ICMP 18581* Coffea arabica Thailand JX010165 FJ907426 C. gloeosporioides IMI 356878* Citrus Italy JX010152 JX009531 ICMP 17821 C. gloeosporioides CORCG4 Orchis sp. China HM034808 HM034800 C. gloeosporioides CORCG5 Orchis sp. China HM034809 HM034801 C. horii NBRC 7478* Diospyros kaki Japan GQ329690 JX009438 ICMP 10492 C. kahawae IMI 319418* Coffea arabica Kenya JX010231 JX009452 C. musae CBS 116870* Musa sp USA JX010146 JX009433 ICMP 19,119 C. nupharicola CBS 470.96* Nuphar lutea subsp. polysepala USA JX010189 JX009486 ICMP 18187 C. psidii CBS 145.29* Psidium sp Italy JX010219 JX009515 ICMP 19120 C. queenslandicum ICMP 1778* Carica papaya Australia JX010276 JX009447 C. salsolae ICMP 19051* Salsola tragus Hungary JX010242 JX009562 C. siamense ICMP 18578* Coffea arabica Thailand JX010171 JX907423 CBS 130417 C. siamense BMLI15 Coffea arabica Thailand FJ972614 FJ907422 C. siamense ALSKN-CG1 Mangifera indica Ghana MT450687 MT452577 C. siamense ALSKN-CG2 Mangifera indica Ghana MT450688 MT452578 C. siamense ALSKN-CG3 Mangifera indica Ghana MT450689 MT452579 C. siamense ALSKN-CG4 Mangifera indica Ghana MT450690 MT452580 C. siamense ALSKN-CG5 Mangifera indica Ghana MT450691 MT452581 C. theobromicola CBS 124945* Theobroma cacao Panama JX010294 JX009444 ICMP 18649 C. ti ICMP 4832* Cordyline sp. New Zealand JX010269 JX009420 C. trichellum CBS 217.64 Hedera helix GU227812 GU227970 C. xanthorrhoea BRIP 45094* Xanthorrhoeae preissii Australia JX010261 JX009478 ICMP 17903 *Type strain. Isolates with names in bold print were obtained in this study 1 3 Indian Phytopathology alignment generated, using MEGA5 (Tamura et al. 2011). disease incidence after which a sub-sample of 50 were Clade stability of the tree resulting from maximum parsi- picked at random for disease severity assessment. Disease mony analysis was assessed by bootstrap analysis with 1000 incidence and severity were calculated as described earlier replicates (Felsenstein 1985). in this work. After that, the fruits from the three trees of each replicate were bulked and separated into exportable, market- Effect of selected copper based fungicides able and non-marketable fruits based on the percentage of on the incidence and severity of tear stain disease the surface area covered by the disease symptoms. Percent- age exportable fruits was calculated using the formula: Two copper based fungicides, one synthetic inorganic fungi- cide and Bion (Acibenzolar-s-methyl), a biostimulant were number of fruits that can be exported Percentage exportable fruits = × 100 Total number of fruits procured from commercial pesticide shops and evaluated on Data on disease incidence and percentage exportable a 9 year old mango orchard, present at the Soil and Irriga- fruits were arcsine transformed and subjected to analysis of tion Research Centre of the University of Ghana, located at variance (ANOVA). On the other hand, ANOVA was per- Kpong, in the Eastern Region of Ghana. The experiment was formed directly on severity indices. Means were separated carried out in the major and minor seasons of 2020, on the using LSD at 5%. Keitt variety of mango. Six treatments were evaluated in the study these were; cuprous oxide (750 g/kg) at 2 gl−1; copper oxychloride (500 g/kg) at 3 gl−1; Mancozeb (800 g/kg) at 4 −1 Resultsg l ; cuprous oxide (750 g/kg) + acibenzolar-S-methyl at 2 gl−1 + 0.17 gl−1, copper oxychloride (500 g/kg) + acibenzo- Disease prevalence, incidence and severity lar-S-methyl at 3 gl−1 + 0.17 gl−1 and non-treatment con- in different mango growing districts of Ghana trol. Prior to each experiment, excess leaves, twigs and fruits from the previous seasons’ production were pruned off and The tear stain disease symptoms were prevalent in the study removed from the orchard. Trees were induced to flower area. In the major season, the prevalence ranged from 50% using potassium nitrate. Trees with uniform flowering were in the Asutuare and Juapong areas to 70% in Akuse, in the selected and the treatments were applied using a mistblower. major season. The prevalence was however, higher in the A randomised complete design with four replicates was used major season, with the disease being found in all farms in the trial. Each replicate was made up of three trees. Treat- inspected in the minor season (Table 2). The disease inci- ments were applied at bi-weekly intervals, beginning from dence also ranged from 9.4% in Somanya to the highest of when more than 50% fruit set was observed, and ended at 14.4% in Akuse in the major season. During the minor sea- 7 days before physiological fruit maturity. In all, there were son, the disease incidence ranged from 37.3% to 45.7% in 5 applications of fungicides. Seven days after the last treat- the study area, with the highest being observed in Dodowa ment application, all the fruits on each tree were harvested (Table 2). Disease severity also ranged from 0.08 (Somanya) and 200 were selected at random and used to determine Table 2 Prevalence of mango Area Prevalence Incidence Severity tear stain disease and the (%) π ± se (%) π ± se π ± se disease incidence and severity in the indicated areas in the Major season Minor season Major season Minor season Major season Minor season major and minor season of 2019 Akuse 70.0 100.0 14.4 ± 0.5 40.9 ± 0.9 0.16 ± 0.01 1.98 ± 0.06 Asutuare 50.0 100.0 9.7 ± 0.2 44.4 ± 1.3 0.13 ± 0.03 1.4 ± 0.06 Dodowa 60.0 100.0 9.9 ± 0.3 45.7 ± 0.1 0.1 ± 0.01 1.5 ± 0.7 Kpong 40.0 100.0 11.9 ± 0.4 42.9 ± 0.9 0.1 ± 0.01 2.08 ± 0.06 Juapong 50.0 100.0 11.5 ± 0.5 42.9 ± 0.6 0.1 ± 20.001 1.6 ± 0.07 Somanya 60.0 100.0 9.4 ± 0.3 37.3 ± 10.5 0.08 ± 0.01 2.1 ± 0.06 π ± se = means + standard error 1 3 Indian Phytopathology to 0.16 (Akuse) in the major season while it ranged from 1.4 and hyaline spores (Fig. 1e). The spore sizes were 15.3 µm in Asutuare to 2.1 in Somaya in the minor season (Table 2). to 16.1 µm long while the width was from 4.9 µm to 5.3 µm. The appressorial length was 8.9 µm to 9.0 µm with width of Cultural and Morphological characterisation 4.8 to 7.0 µm. The growth rate of 1.10 mm/day, was same of the isolated fungi for all isolates (Table 3). Isolates of the fungus showed similar cultural characteristics. Pathogenicity test of isolates They produced white mycelium which grew to fill an entire 8 mm plate in 7 days (Fig. 1c). After 7 days, numerous bright All isolates were able to induce anthracnose disease symp- orange coloured acervuli, which darkened with time, were toms on the inoculated fruits surfaces. The symptoms on the found in concentric rings in the middle of the culture. The fruits began as small dark brown spots which were slightly disc-shaped acervuli had numerous black setae on their sur- sunken. The spots expanded and covered large areas of the faces (Fig. 1d) and burst to release numerous short conical fruits surface by the 7th day after incubation (Fig. 1f). The same fungi were re-isolated from the induced symptoms. Table 3 Conidia and Isolate Designation aMean conidium size aMean appressorium size Growth rate appressoria size and (µm) (µm) (mm/day) pathogenicity of Colletotrichum isolates from tear stain ALSKN-CG1 15.9 ± 0.3 × 4.9 ± 0.1 9.2 ± 0.1 × 5.5 ± 0.1 1.10 symptoms on mango fruits ALSKN-CG2 16.0 ± 0.3 × 4.9 ± 0.3 8.9 ± 0.2 × 4.8 ± 0.1 1.10 ALSKN-CG3 15.8 ± 0.1 × 4.7 ± 0.3 8.6 ± 0.2 × 5.7 ± 0.2 1.10 ALSKN-CG4 16.0 ± 0.2 × 4.3 ± 0.2 8.9 ± 0.1 × 5.3 ± 0.3 1.10 ALSKN-CG5 16.0 ± 0.3 × 4.7 ± 0.3 8.9 ± 0.1 × 5.0 ± 0.1 1.10 a Mean ± s.e Fig. 2 Maximum parsimony phylogram constructed from the multiple sequence alignment of the combined nucleotide sequences of the ITS region and the ACT gene. Colletotri- chum trichellum was used as outgroup. With the exception of strains whose names were preceded by ALSKN, all strains are either isolates of confirmed identities (names in Bold) or are ex-type strains. Sequences of ex-type strains or strains with confirmed identities were downloaded from the EMBL database 1 3 Indian Phytopathology Molecular characterisation was significant difference (p > 0.05) in percentage of export- able fruits on trees that received the different treatments. The Analysis of the sequences and phylogenetic studies highest was obtained on trees treated with Mancozeb while of the ITS region and the partial actin gene the lowest was obtained on control trees. The percentage of clean fruits on trees treated with Mancozeb was the same as The size of the amplified PCR product of the ITS region what was obtained on trees treated with all other fungicides and of the partial actin gene obtained from the isolates were except Nordox (Table 4). approximately 600 bp and 250 bp respectively. After assem- Results obtained in the minor season were similar to what bling, sequences obtained were 535 bp and 241 bp long for was observed in the minor season. The significantly high- the ITS region and ACT gene, respectively. The most par- est disease incidence and severity were obtained on trees simonious tree obtained using the concatenated sequences that were not treated with any fungicide while the lowest of the two genes is shown as Fig. 2. The tree length was was obtained on trees that received one of the fungicidal 166, the consistency, retention and composite indices were treatments. On the other hand, the lowest percentage of respectively, 0.602740, 0.764228 and 0.630718 (0.460630) exportable fruits was obtained on trees that did not receive for all sites and parsimony-informative sites (in parenthe- any treatment while all fungicidal treatments resulted in the ses). Shown next to the branches are the bootstrap values same percentage of exportable fruits (Table 4). in percentages. There were 631 sites in the final data set. The different isolates clustered in different clades, with all isolates obtained from the tear stain symptoms on mango, Discussion clustering in a clade containing the C. siamense type strain and the other C. siamense isolates whose identities are well The tear stains symptoms on mango fruits in Ghana has known. The clade was supported with a high bootstrap value been a subject of many discussions. While some people are of more than 70% (Fig. 2). of the opinion that the symptoms were healing scars from wound created by unknown causes, very few were of the Effect of fungicides on disease incidence and severity view that they represent another form of the anthracnose and percentage of exportable fruits of mango. symptom. Findings from this research work have shown that the symptoms were caused by known causal agents of There was significant difference (p > 0.05) in the incidence anthracnose and hence the symptoms were another form of of the tear stain disease on mango fruits in the major season the anthracnose disease. Typically, anthracnose refers to a of 2019. The highest incidence was observed on trees that dark brown spot which is slightly sunken (Agrios 2005) and did not receive ant fungicidal treatment while the lowest has been associated with members of the genus Colletotri- was observed on trees treated with a combination of cuprous chum (Ploetz 1998; Arauz 2000). This description is very oxide and acibenzolar-S-methyl (Table 4). The difference different from what was described for the tear stain. In fact in in disease incidence on all trees that received fungicidal this study, the tear stain symptoms were blister-like spots on treatments, was however, not significant. Similarly, the dis- the infected fruit surface and are rough to touch. However, ease severity was highest on trees that were not treated with when infected fruits were incubated for some time, it was any fungicide while the lowest was on trees treated with observed that the typical dark brown symptoms of anthrac- cuprous oxide and acibenzolar-S-methyl. However, there nose began to radiate out from the dried tear stain spots. This was no significant difference in disease severity among fun- gives an indication that the tear stain spots were anthracnose gicidal treatments evaluated in the study (Table 4). There symptoms that stopped expanding and eventually developed Table 4 Effect of fungicides Treatments Major season Minor season on the disease incidence and severity and percentage of Incidence Severity Exportable Incidence Severity Exportable exportable mango fruits in the fruits (%) fruits (%) major and minor season of 2020 Curenox 4.2a 0.01a 95.3b 5.8a 0.12a 85.9b Nordox 4.1a 0.01a 87.6bc 4.1a 0.10a 90.9b Curenox + Bion 5.6a 0.02a 93.0bc 6.1a 0.12a 83.5b Nordox + Bion 2.9a 0.01a 94.4bc 2.9a 0.13a 87.7b Mancozeb 5.0a 0.01a 95.5bc 5.0a 0.13a 84.3b Control 31.8c 1.20b 38.4a 30.3b 1.90b 28.9a Means followed by same alphabets in a column are not significantly different 1 3 Indian Phytopathology dried blisters in the field and which then resumes expansion (Phoulivong et al 2010; Abang 2003). In Ghana, the ITS after harvest when right conditions for the expansion occur region has been combined with other gene such as the beta in storage. These observations confirm the reports by Nelson tubulin and glyceraldehyde-3-dehydrogenase gene to differ- (2008) and Kumar et al (2016) that the tear stain symptoms entiate among species within the C. gloeosporioides com- were another type of anthracnose. plex (Honger et al. 2014; 2016). Phylogenetic studies involv- The disease was found to be very prevalent in the study ing the nucleotide sequences of the ITS region and partial area, both in the major and minor seasons. Since blemished actin gene showed that the causal agent of the tear stain fruits are considered unfit for the international markets disease in Ghana was C. siamense. The gene tree drawn with (Arauz 2000) the disease remains a major threat to the prof- the combined sequences of the two gene regions resulted itability of mango farmers in the study area. The disease in the clustering of the isolates with the type strain of C. incidence and severity also appeared to be higher in minor siamense in a clade supported by a high bootstrap support. season than in the major season, confirming the reports by In the same phylogram, the clade made up of type strain of farmers. Naturally, fungal disease prevalence correlated C. gloeosporioides and other C. gloeosporioides strains of positively with environmental factors (Arauz 2000; Agrios confirmed identities did not include any of the isolates from 2005), therefore, the high humidity and rainfall that pertains the tear stain symptoms in the study. This confirms that the in the major season compared to the minor season in the isolates were C. siamense and not C. gloeosporioides. Simi- study area, would have exacerbated the disease in the for- lar results have been reported in Ghana (Honger et al. 2014). mer than the latter season (Arauz 2000). On the other hand, C. siamense has been reported causing the typical the short period between the major and the minor season sunken spots characteristic of anthracnose on mango in in these areas, makes it impossible for farmers to carry out Ghana (Honger et al. 2014). However, though the fungus proper cultural practices such as pruning of trees and clean- was isolated from fruits showing the tear stain symptoms ing of farms, prior to the minor season. Since the disease is in this study, it could not induce the same symptom on the caused by a fungus, poor tree pruning and farm sanitation inoculated fruits. This could be due to the absence of cer- activities will enhance its proliferation. This could account tain factors which may be restricted to the field. In fact, in for the higher disease incidence in the minor season than in Ghana, there is higher incidence of tears stain infection in the major season. Tear stain disease therefore, remains an the minor mango season that the major season, which show important disease of concern as it has the potential of reduc- varied weather conditions (Honger: unpublished data), giv- ing the quantities of mangoes exported to the international ing credence to the suggestion that field factors including markets from the study area, which remains an important weather conditions could be involved in the development of source of exportable mangoes from Ghana. the symptoms in the field. However, by inducing the slightly While the nature of the tear stain symptom indicates that sunken dark spots in this study, it confirms the fungus as it was another type of anthracnose symptom, identifying pathogenic to mango. the causal agent was found to be important in clearing all Application of synthetic chemicals, both from organic doubts about its identity. Elsewhere, the tear stain disease and inorganic sources has been employed for the control symptoms on mango fruits have been attributed to C. gloe- of mango anthracnose disease in several parts of the world osporioides (Kumar et al. 2016; Nelson 2008). In this study, (McMillan 1984; Dirou and Stovold 2005; Honger 2013; the fungus consistently isolated from the disease symptoms Nasir et al. 2017). In this study, two copper based fungicides produced short conical spores with rounded edges in disc and mancozeb were found to be very effective in reducing shaped acervuli with setae. Isolates of the fungus caused the incidence of the disease. Mancozeb is permitted to be the typical anthracnose symptoms on the inoculated mango used on mango fruits that are exported to European countries fruits. These morphological characteristics of the fungus and where some of the mangoes from Ghana are sent (Arauz, its pathogenicity on the crop confirmed the causal agent as 2000). On the other hand, copper based fungicides are C. gloeosporioides (Honger et al. 2014; Arauz 2000; Damm acceptable in all markets, including the organic markets. et al. 2010). The effectiveness of these fungicides in this study, therefore, Colletotrichum gloeosporioides is a group species made gives options for mango farmers to reduce the effect of the up of several distinct species sharing common cultural and disease on their fruits for export. Recently, the possibility of morphological features (Damm et al. 2010). Even, the vari- boosting the natural resistance of susceptible fruits against ability of the sequences of the ITS region, which has been anthracnose disease, has been investigated by the applica- used widely as a bar code for species delineation in fungi, tion of salicylic acid and acibenzolar-S-methyl (Zainuri has been found to be inadequate in separating among spe- et al. 2003). In this study, Bion (acibenzolar-s-methyl), was cies in the C. gloeosporioides complex (Weir et al. 2012). combined with the fungicides to boost their performance. To be able to achieve this, a polyphasic approach which Though, the effect of the biostimulant was not felt, (as the relies on the use of more than one region has been proposed fungicides used without the biostimulant gave same results 1 3 Indian Phytopathology as when it was used), it’s possible that the results may be Carbone I, Kohn LM (1999) A method for designing primer sets for spe- felt, with longer use. ciation studies in filamentous ascomycetes. Mycologia 91:553–556 Damm U, Baroncelli R, Lei C, Kubo Y, O’Donnell R, Weir B, Yoshino K, Cannon PF (2010) Colletotrichum: species, ecology and interac- tions. Int Mycol Assoc 1(2):161–165 Conclusion Dirou J, Stovold G (2005) Fungicide management program to control mango anthracnose. Primefact 19. Department of Primary Indus- Tear stain disease of mango fruits was found to be preva- tries, New South Wales (ISSN 0725-7759)E. P. A (2015) List of registered products in Ghana. Environmental Pro- lent in the coastal savannah area of Ghana, where the high- tection Agency, Accra est concentration of mango farms are found in the country. Felsenstein J (1985) Confidence limits on phylogenies: an approach using More of the disease was found in the minor season than in the bootstrap. Evolution 39:783–791 the major season and this has been attributed to poor cul- Honger JO (2013) Characterisation of the causal agent of mango anthrac-nose disease in Ghana. Doctoral Thesis, Department of Crop Sci- tural practices in mango orchards in the minor season. The ence, University of Ghana causal agent of the tear stain symptoms on mango fruits Honger JO, Offei SK, Oduro KA, Odamtten GT, Tatu SN (2014) Identi- was identified as C. siamense based on the traditional meth- fication and species status of the mango-biotype of Colletotrichum ods and sequence analysis of the ITS region and the actin gloeosporioides in Ghana. Eur J Plant Pathol 140:455–467Honger JO, Offei SK, Oduro KA, Odamtten GT, Tatu SN (2016) Iden- gene. The pathogen was able to induce the typical dark tification and molecular characterisation of Colletotrichum species brown sunken spots associated with anthracnose disease on from avocado, citrus and pawpaw in Ghana. South African J Plant mango fruit, but not the tears stain symptoms on artificially Soil 33(3):177–185. https://d oi.o rg/1 0.1 080/0 25718 62.2 015.1 1259 inoculated fruits. Nonetheless, the identity of the pathogen 58Johnston PR, Jones D (1997) Relationship among Colletotrichum iso- and it’s pathogenicity on mango fruits confirmed that the lates from fruit rots assessed using rDNA sequences. Mycologia tear stain disease was another symptom of anthracnose and 89(3):420–430 hence could be controlled using the same strategy employed Kumar P, Kumar GV, Kumar TA (eds) (2016) Current trends in plant dis- to control the well-known anthracnose disease symptom. ease diagnostics and management practices. Springer international publishing, Switzerland Consequently, three fungicides, namely, Mancozeb, copper Lakshmi BKM, Reddy PN, Prasad RD (2011) Cross infection potential oxide and copper oxychloride were found to be very effec- of Colletotrichum gloeosporioides Penz, isolates causing anthrac- tive against the disease. A combination of the copper based nose in sub-tropical crops. Trop Agricult Res 2:183–193 fungicides with Bion (Acibenzolar-S-methyl), a biostimu- McMillan RT (1984) Control of mango anthracnose with foliar sprays. Proc Florida State Horticult Soc 97:344–345 lant, also controlled the disease very well. Findings from this MOAP (2016) Post-harvest losses in the mango, pineapple and citrus research have provided enough information for the control of value chains in Ghana. In: Data provided by GIZ the disease in the study area and these must be implemented. Nasir M, Iqbal B, Idrees M, Sajjad M, Niaz MZ, Anwar H, Shehzad MA, Tariq AH (2017) Efficacy of some organic fungicides against anthracnose and powdery mildew of mango. Pakistan J Agricult Sci 54(3):493–496 Funding NA. Nelson SC (2008) Mango anthracnose (Colletotrichum gloeosporioides). College of Tropical Agriculture and Human Resource. Publication Availability of data and material NA. 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Plant Dise 84(6):600–611 1 3 Indian Phytopathology Zainuri Z, Irving DE, Dann EK, Coates LM, Wearing AH (2003) Activat- Publisher's Note Springer Nature remains neutral with regard to ing mango fruit defense to anthracnose disease. In: Proceedings of jurisdictional claims in published maps and institutional affiliations. Australian postharvest horticulture conference, Brisbane, Queens- land, pp 149–150 1 3