Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 113-124

ISSN: 2319-7706 Volume 3 Number 4 (2014) pp. 113-124 http://www.ijcmas.com

Review Article Citrus Melanose (Diaporthe citri Wolf): A Review

K.Gopal*, L. Mukunda Lakshmi, G. Sarada, T. Nagalakshmi, T. Gouri Sankar, V. Gopi and K.T.V. Ramana

Dr. Y.S.R. Horticultural University, Citrus Research Station, Tirupati-517502, Andhra Pradesh, India *Corresponding author

A B S T R A C T

K e y w o r d s Citrus Melanose disease caused by Diaporthe citri Wolf is a fungus that causes two distinct diseases on Citrus species viz, the perfect stage of the fungus causes Citrus melanose, disease characterized by lesions on fruit and foliage and in the imperfect Melanose; stage; it causes Phomopsis stem-end rot, a post-harvest disease. It is one of the Diaporthe most commonly observed diseases of citrus worldwide. As the disease is occurring citri; in larger proportions and reducing marketable fruit yield hence, updated post-harvest information on its history of occurrence, disease distribution and its impact, disease pathogen and its morphology, disease symptoms, epidemiology and management are briefly reviewed in this paper.

Introduction

Citrus Melanose occurs in many citrus fungus does not normally affect the pulp. growing regions of the world and infects On leaves, the small, black, raised lesions many citrus species. It affects young are often surrounded by yellow halos and leaves and fruits of certain citrus species can cause leaf distortion. On the fruit, the or varieties when the tissues grow and disease produces a superficial blemish expand during extended periods of rainy which is unlikely to affect the overall yield or humid weather conditions. The of processing fruit, but causes external symptoms of this widely distributed fungal blemishes which reduce the value of fruit disease vary from small spots or scab-like intended for the fresh market. This disease lesions to patterns of damage referred to as is generally of minor economic importance tear-drop, mudcake, and star melanose. on foliage. All commercial citrus varieties This is one of the most commonly grown in Florida are susceptible to observed diseases of citrus fruits melanose. Pustules are larger on worldwide. Citrus melanose is caused by grapefruit, so blemishes tend to be more the plant-pathogenic fungus Diaporthe serious on this species. The disease citri (anamorph = Phomopsis citri). It can typically attacks sweet orange (Citrus create severe fruit rind blemishes, but the sinensis), grapefruit (C. paradisi) and

113 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 113-124 pummelo (C. grandis). The disease inoculum, and from pure cultures of P. reduces the aesthetic quality of fresh fruits citri both melanose and stem-end rot could for the market, although it does not affect be reproduced on citrus plants. Fawett edibility. (1932) compared isolates of P. citri and D. citri from across the globe as well as P. History, Taxonomy and Phylogeny californica and found the teleomorphs indistinguishable. Eight years later Ruehle Diaporthe citri Wolf and Kuntz (1940) showed that the spores (anamorph: Phomopsis citri Fawc.) is a of both P. citri and D. citri caused fungus that causes two distinct diseases melanose on citrus leaves. on Citrus species. In the perfect stage, this fungus causes melanose, disease Because the name Diaporthe citri has been characterized by superficial fruit and foliar applied to melanose and stem rot disease lesions, and in the imperfect stage, it of Citrus for decades and Fawcett (l.c.) causes Phomopsis stem-end rot, a post- explicitly stated that he was unaware of harvest disease (Whiteside, 1988). While any previous Diaporthe or Phomopsis on melanose was first found and described by Citrus, it is proposed that Phomopsis citri Swingle and Webber in 1892 near Citra, H.S. Fawc. be conserved against its earlier Florida (Wolf, 1926), it wasn t until 1928 homonym P. citri (Sacc.) Traverso & that Koch s postulates were completed Spessa.This will make the former name and Diaporthe citri was confirmed as the available as a basionym for Diaporthe citri causal agent of melanose on citrus (Bach (H.S. Fawc.) F.A. Wolf, giving this name and Wolf, 1928). In 1912, Fawcett priority over what would otherwise be the proposed the causal agent of stem-end rot earlier taxonomic synonyms D. citrincola as P. citri. Descriptions of two pathogens and Phomopsis caribaea, both of which belonging to the genus Phomopsis that predate the currently first legitimate caused stem-end rot on citrus were publication as Diaporthe citri F.A. Wolf. published in 1922, and they were given As no type material of P. citri was found different species names because of at either BPI or FLAS, leaving only an differences in growth characteristics in illustration (Fawcett, l.c.) as a potential, vitro and pathogenicity in planta. The first but unsatisfactory, iconotype, a recent was discovered in the Isle of Pines, West specimen from diseased Citrus sp. Hence, Indies, in 1917, on grape fruit, and the it is proposed as a conserved type for name P. caribaea was proposed by the Phomopsis citri (Amy Rossman et al author (Horne, 1922). The second was 2013). found in 1919 in California on lemons. The fungus was tentatively named P. Another synonymous species of D. californica (Fawcett, 1922).The first citri is D. medusaea Nitschke, and because evidence that these two diseases melanose the latter species was described first, and stem-end rot - were caused by the Fisher proposed in 1972 that the name D. same fungus was published by Floyd and medusaea should take precedence over D. Stevens in 1912. The authors were unable citri. In the same article, Fisher proposed to isolate any fungus from melanose that because P. cytosporella was described infected leaves, but could reproduce prior to P. citri, the former name should be classic symptoms of melanose by using used. This nomenclature was never washings of stem-end rot affected twigs as adopted. The genus Diaporthe/ Phomopsis

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Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 113-124 belongs to the phylum Ascomycota, tef1- , tubulin and calmodulin grouped class Sordariomycetes, order Diaporthales, eight most discrepant strains into three and family Diaporthaceae (James et al. distinctive clusters, cluster 1 (Rc001, 2006; Rossman et al. 2007). Combining Eu004 and Eu009), cluster 2 (ZJWCF252, morphological, cultural and molecular Sjm001 and Ac001) and cluster 3 (Pcs013 data, four species were distinguished from and Sfp005) respectively with high F. vulgare. Diaporthe angelicae is shown support values. This cluster above to be the most common pathogen of this represent three potentially novel species host in Portugal. Diaporthe lusitanicae is provides strong evidence of high newly described, whereas the teleomorph biodiversity and novelty of Phomopsis theicola was revealed to be of Diaporthe endophytes from southeast distinct from Diaporthe theicola, and is China (Jiaying Wang et al 2014). Six new described as Diaporthe neotheicola species of Diaporthe, D. beilharziae (Santos and Phillips, 2009). To date, on Indigofera australis, D. fraxini- several molecular phylogenetic studies on angustifoliae on Fraxinus angustifolia genera within the Diaporthales have been subsp. oxycarpa, D. litchicola on Litchi published (Zhang and Blackwell, 2001; chinensis, D. nothofagi on Nothofagus Castlebury et al. 2002; Udayanga et al. cunninghamii, D. pascoei on Persea 2011; Udayanga et al. 2012) but none americana and D. salicicola on Salix have included D. citri/P. citri. Among the purpurea from Australia are described and eight described families within illustrated based on morphological the Diaporthales, the family most related characteristics and molecular analyses to Diaporthaceae is likely Valsaceae (Tan et al; 2013). (Castlebury et al. 2002). Although species of Diaporthe have in the past chiefly been Symptoms distinguished based on host association, Gomes et al (2013) studies confirm Melanose on Leaves: Symptoms on several taxa to have wide host ranges, foliage begin as tiny water-soaked specks suggesting that they move freely among that become depressed in the center and hosts, frequently co-colonising diseased or surrounded by a translucent yellow area dead tissue. In contrast, some plant which is not depressed. Later, the leaf pathogenic and endophytic taxa appear to cuticle ruptures and a gummy substance is be strictly host specific. Given this diverse exuded which turns brown and hardens. ecological behaviour among members of The yellowish margin disappears and the Diaporthe, future species descriptions hardened gummed areas will have a lacking molecular data should be strongly sandpaper-like texture. Infected areas on discouraged (Gomes et al. 2013). Multi- the leaf may be scattered, aggregated, or in locus phylogeny based on combined streaks, depending on where water sequences of rDNA ITS, and partial transported the inoculums prior to sequences from the translation elongation infection. Severely infected leaves become factor 1- , tubulin and calmodulin pale green to yellow, can be distorted and genes, reveal three new species, Diaporthe fall from the tree. Melanose is seldom siamensis, D. thunbergii and D. severe on the spring growth flush. When it pterocarpicola from Thailand (Dhanushka does occur on this flush, the pustules are Udayanga et al; 2012). Multi-gene usually few and little or no leaf drop phylogenetic analysis based on ITS rDNA, occurs (Fig. 1). On the summer growth

115 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 113-124 flushes, melanose can be severe enough to about 80 countries. Further, seven of the cause defoliation, particularly in years ten highest citrus producing countries are following freeze-induced twig die-back. reported to have D. citri (FAOSTAT, On fruit there is a tendency for the 2010). Fruit severely affected by melanose diseased areas to form tear-streak and is not marketable as fresh fruit and water droplet patterns. Young green twigs therefore end up being processed for can be infected. In most cases, it is juicing. This severely reduces market difficult and not economical to try to value to 1/5 of the value relative to that of control melanose on the foliage. non-blemished fruit. Slightly affected fruit can still be sold in the fresh fruit market, Melanose on Fruit but the value is ¼ that of non-blemished fruit (Kuhara, 1999). This loss of value Light infestation produces scattered specks can cause significant impacts on farmers on the fruit. Fruit infected when young and fruit exporters. Melanose affects all may remain small and abscise citrus species, but grapefruit and lemons prematurely. Late infection produces are the most susceptible. The disease flatter pustules. Under severe condition occurs in most citrus-growing areas of the solid patches of blemish are produced, the world. It is most severe in moist, fruit surface can crack producing a subtropical regions, less severe in the roughened condition called mudcake humid tropics, and unimportant in arid melanose. Mudcake melanose occurs if areas. It usually does not affect tree infection takes place soon after petal fall growth or fruit yield, but can reduce (Fig. 1). Melanose on fruit can be profitability of the fresh-market fruit. In distinguished from rust mite injury by the Florida, Timmer et al. (1998) reported that presence of a roughened surface with a reduction of 10% in the percentage of melanose and a smooth rind blemish with fresh-market grapefruit by melanose rust mite injury. caused a loss of $866 per hectare.

Star Melanose The pathogen and morphology

Star melanose occurs when copper is The fungal pathogen Diaporthe citri Wolf applied late during hot, dry weather and is is the teleomorph or sexual stage, and the due to copper damage to leaves. It has no anamorph is Phomopsis citri Fawc. A relationship to melanose but may resemble synonym for D. citri is D. medusaea Nits. symptoms of that disease. Copper causes The pathogen mostly infects leaves and the developing tissues to become more fruits, but it may also cause a stem-end rot corky and darker than normal and the of citrus, and it infests dead twigs as a shape of the lesion often resembles a star saprophyte. (Fig. 1). D. citri produces two-celled hyaline Distribution and impact ascospores, and within each cell are two oil droplets, or guttulae. Ascospores may D. citri affects all citrus cultivars, although be slightly constricted at the septum and it is most severe in grapefruit and lemons range in size from 11.5 to 14.2 microns by (Mondal et al. 2007). The disease is 3.2 to 4.5 microns (Fig. 2). Ascospores are present in most citrus growing areas in borne inside the perithecium, a flask

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Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 113-124 shaped structure. The perithecia are forming mycelium on a new substrate and circular, flattened at the base, with long starting the disease cycle over again black beaks that taper out. The beaks (Whiteside, 1988; Wolf, 1926). range from 200 to 800 microns long by 40 to 60 microns wide, and the base of the The fungal mycelia are never abundant in perithecium typical size ranges from 125 melanose lesions. The production of gum, to 160 microns in diameter (Whiteside, sparse amounts of mycelia in melanose 1988; Wolf, 1926). The perithecial lesions and the ubiquitous presence stromata stay within the bark of the plant, of Colletotrichum gloeosporioides on the but the perithecia protrude to the outer surface of most Citrus leaves and surface of the stem and are plainly visible secondary fungal invaders in the fissures with a dissecting microscope. Because created by D. citri, caused significant ascospores are forcibly ejected from the delays in fulfilling Koch s postulates early asci, they become windborne and are in the course of the research on D. responsible for the long distance spread of citri (Bach and Wolf, 1928; Whiteside, the pathogen (Wolf, 1926). Upon finding a 1988). Fifteen new species are described, suitable substrate, one or both spores may namely Diaporthe arengae, germinate, producing hyphae that quickly D. brasiliensis, D. endophytica, become septate mycelium (Whiteside, D. hongkongensis, D. inconspicua, 1988). In culture, the mycelium is fan- D. infecunda, D. mayteni, D. neoarctii, D. shaped and white in color (Timmer et oxe, D. paranensis, D. pseudomangiferae, al.2004). D. pseudophoenicicola, D. raonikayaporum, D. schini and D. The conidial state of the fungus is the most terebinthifolii. A further 14 new important for the disease cycle. P. combinations are introduced in Diaporthe, citri produces copious in pycnidia. The and D. anacardii is epitypified (Gomes et pycnidia are scattered on the substratum al. 2013). and are dark in color, ovoid, thick-walled and erumpent. Pycnidia form on dead Epidemiology wood and decaying fruit. The fungus produces two types of conidia: alpha and Susceptibility of citrus tissue beta. The alpha conidia are functional, single-celled, hyaline, and contain two Melanose attacks the foliage, fruit and guttulae. They are the primary means by twigs when they are immature. As the which the fungus is disseminated short tissues mature they become more resistant distances, and they range in size from 5 to to infection. Immediately after petal-fall 9 microns by 2.5 to 4 microns. The beta the young developing fruit are very conidia tend to be produced in older susceptible to infection and if wet pycnidia than the alpha conidia, and are conditions prevail for several days at this long, slender rod shaped structures that do stage, serious blemishes on fruit will not germinate and are hooked at one end. occur. Fruit rind becomes more resistant to Because the conidia are embedded in a infection eight or nine weeks after petal slimy matrix within the erumpent fall, no further infection of fruit. The pycnidium, rainy conditions spread the symptoms of melanose on fruit can differ conidia to nearby substrates with great according to the time and level of efficiency. The conidia then germinate, infection. Mud cake melanose develops

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Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 113-124 when the rind is heavily infected shortly ambient temperature and moisture on the after petal fall, mostly on late bloom fruit. surface, in approximately 36 to 48 hours Infection towards the end of the period of the spores will germinate, and a germ tube susceptibility produces small spots will directly penetrate the cuticle layer referred to as flyspeck melanose (Bach and Wolf, 1928). The hyphae (Whiteside, 1976). A leaf remains branch out and travel between epidermal susceptible to melanose infection for only cells. The cells are degraded by enzymatic about a week after unfolding from the bud. action, and hemicelluloses gum surrounds Although immature leaf growth can be the infected tissue and forces parenchyma infected in the early spring it is not usual cells apart. At this point, typically 4-5 days for melanose to be active in August to post inoculation, the degradation of cells September. However, the late summer leaf becomes visible as a sunken area on the flush is often badly infected. Serious leaf surface. As the infection progresses, defoliation and death of shoots can the plants wound response catalyzes the adversely affect the productivity of trees. formation of a suberized layer of corky This applies particularly to Washington tissue, which grows until it reaches 7 to 12 navel oranges. Disease severity is greatly layers of individual cells. The gum affected by tree vigour. Young vigorous darkens while the normal cells beneath trees need precautionary spraying in early continue growing. This corky layer is years. pushed out, finally bursting the cuticle, creating small, brown pustules that have D. citri is primarily a saprophyte that the feel of sandpaper. survives on and derives energy from living and dead wood (Kucharek et al. 2000; Disease Cycle Mondal et al. 2007). Perithecia and pycnidia are only produced on dead and Recently killed young twigs in the tree dying twigs and on fruit affected by stem- canopy supports spore production for end rot, and because perithecia are rarely occurrence of fungal infection. Also, the formed, the conidia produced by pycnidia host tissue must be in a susceptible state. are the principal source of inoculum (Bach Temperature and rainfall conditions during and Wolf, 1928; Kuhara, 1999). the period of leaf expansion and during the Ascospores are forcibly discharged and are first 12 weeks after petal fall regulate important for long distance dispersal disease severity. After a spore lands on (Kucharek et al.2000). susceptible tissue, a period of 10-12 hours of moisture is required for infection at Infection of the disease is initiated when 25°C whereas at 15°C 18-24 hours of ascospores of the Diaporthe stage or wetness are necessary for infection. Thus conidia of the Phomopsis stage land on extended wet periods resulting from leaf tissue. In dry conditions, or at afternoon rain showers plus dew periods in temperatures below 17°C or above 35°C, May and June coupled with warmer the spores die and no infection occurs temperatures during these months create (Kuhara, 1999). It has been found that the favorable weather for infection. In pathogen needs 10 to 24 hours of moisture contrast, rainfall prior to May in central for the spores to germinate, depending on and south Florida often is associated with the temperature (Agostini et al. 2003; fast-moving cold fronts that are quickly Kucharek et al. 2000). Given sufficient followed by temperatures that are too cool

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Fig.1 A: Symptoms of Melanose pustules on grapefruit leaf and fruit; B: Speck melanose on grapefruit; C: Mudcake and tear-staining symptoms on grape fruit; D: Star Melanose; E: Tear-Stain melanose; F: Melanose symptoms around the stem end of fruit

A B C

D E F

Fig.2A Culture on PDA; B: Conidiophores; C: Conidia

A B C Fig.3 Seasonal development and life cycle of melanose fungus, Diaporthe citri (Harry C. Burnett, 1962).

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Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 113-124 for infection. Also, winds behind the front Disease Control quickly dry surface moisture on foliage. At temperatures between 24-28°C initial Prevention is better than cure, hence symptoms appear 4 to 7 days after sanitation out and removal of dead wood infection. to remove inoculum of the melanose fungus is important, especially in older The fungus Diaporthe citri produces trees. Protectant copper sprays are the only ascospores and pycnidiospores and the product registered for melanose control. latter spore is the part of the life cycle that Timing of spray applications is very provides most of the inoculums for disease important. With Washington navel and (Fig. 3). Ascospores are formed within Valencia oranges the spray should be perithecia on decaying wood on the soil or applied at full petal fall. With lemons on dead branches remaining on the tree. where lemon scab is also a problem, the These spores are produced in relatively initial application should be made at half small numbers and contribute slightly to petal fall. In wet weather, especially if disease potential in a grove. Their main melanose is serious, a follow-up spray contribution to disease development should be applied 6 8 weeks after the relates to spread of the fungus over long initial spray. Whiteside (1985) considered distances because ascospores are it impossible for one treatment to protect windborne. Pycnidiospores, on the other the fruit for the whole 9 12 week period hand, are produced abundantly on dead of fruit susceptibility. If a treatment is branches within pycnidia. They provide applied at petal fall, the fruit will be too for short distance spread within a tree or small to retain much fungicide and what from one tree to an adjacent tree by rain- little material is retained will soon be splash. Spores can be washed down over dissipated through fruit enlargement, if not infected branches by rain or irrigation by rainfall or irrigation. With the petal fall water and spreading them to lower leaves, treatment, only 3 4 weeks protection fruit, or live branch tissue of the tree against melanose can be expected in wet canopy (Fig. 3). conditions, leaving another 6 weeks for the fruit to be attacked. The copper sprays act Therefore, freeze-damaged citrus trees, as protectants, preventing infection of the older groves, and poorly maintained young developing fruit. The melanose groves with much recently killed wood fungus harbored in the dead wood should be considered high melanose throughout the tree is little affected by incidence areas. Leaves become resistant these sprays and they do not reduce the to infection once they are fully expanded. inoculums available from the dead wood. Fruit rind becomes resistant about 12 Where melanose leaf infections are likely weeks after petal fall, but the later the to be serious on foliage in late summer, a infection occurs during that 12- week further protectant copper spray should be period the smaller will be the resulting applied. pustules. Thus, even though suitable weather conditions exist for infection after Grapefruit is susceptible to melanose late June or early July, melanose will not infection from fruit set until it reaches 2.5- infect the fruit rind after that time except 3 inches in diameter, normally in late June in years when the bloom is later than or early July. Fungicides are effective for normal. only short periods when applied to rapidly

120 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 113-124 expanding fruit or leaves. Since April is ying et al 2012) and 24% Fenbuconazole usually a low rainfall month and fruit is SC (QIN Shao-yuan et al; 2012) were the small and growing rapidly, the first spray best one for controlling citrus melanose in for melanose control is not usually applied China. until mid to late April. One or two applications are sufficient for control on The strobilurin fungicides, Abound, Gem, oranges and most tangerines unless the Headline, or Quadris Top, are also trees have abundant dead wood, like in a effective for melanose control and can be year after a freeze. For fresh market used at any time for disease control. grapefruit, the first application should be Copper fungicides are more economical made when the fruit reaches a diameter of and are most important for melanose 1/4 to ½ inch. With average quality copper control. However, since copper fungicides products, usually about 2 lb/acre of applied in hot weather can damage fruit, metallic copper are needed for each 3- use of strobilurins at that time will avoid week period. Rates can be decreased if this damage and control greasy spot as sprayings are given more frequently or well as melanose. Strobilurins appear to increased if sprayings are made less often. have lower residual activity for melanose Additional sprayings should be given at 3- control than do copper fungicides week intervals until the fruit becomes (Dewdney and Timmer, 2011). Thus, resistant. For melanose control on large applications may have to be made at trees, no more than 8-12 lb metallic copper shorter intervals, especially when rainfall is needed per year even if copper is also is high. Further, fungi may develop used for the control of scab and/or greasy resistance to strobilurin fungicides. These spot. Copper residues are reduced with materials should never be used more than fruit expansion and as a result of rainfall. twice in a row, and no more than two It is based on fruit growth models, the rate strobilurin applications should be used for and time of the last application, and melanose control. rainfall since the last spray. It has proven helpful for timing of sprays for melanose References control. An early June spray of copper to control late melanose damage will serve as Amy Rossman., Dhanushka Udayanga., the first greasy spot spray. If copper Lisa, A., Castlebury & Kevin D. Hyde. fungicides are applied from May to 2013. Proposal to conserve the name September, they should be applied when Phomopsis citri H.S. Fawc. (Diaporthe temperatures are moderate (<94°F), at citri), with a conserved type, against rates no more than 2 lb of metallic copper Phomopsis citri (Sacc.) Traverso & per acre, without petroleum oil, and using Spessa (Ascomycota: Diaporthales: spray volumes of at least 125 gal/acre Diaporthaceae). TAXON 62 (3): 627. (Dewdney and Timmer, 2011). Amy Rossman, Y., David, F., Farr Lisa, Applications of pyraclostrobin to the Castlebury, A. 2007.A review of the spring flush growth of citrus trees are phylogeny and biology of the much more likely to provide control of Diaporthales. Mycoscience. (2007) melanose, scab, and Alternaria brown spot 48:135 144. than those of famoxadone or copper Agostini, J.P., Bushong, P.M., A. Bhatia hydroxide (Mondal ,2007). Mancozeb and Timmer, L. W. 2003. Effects of (CHEN Guo-qing et al 2010, JIANG Li- environmental factors on the severity

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