U.S. Department of Agriculture, Agricultural Research Service Systematic Mycology and Microbiology Laboratory - Invasive Fungi Fact Sheets Mal secco disease of Citrus - Phoma tracheiphila Mal secco disease is a vascular pathogen causing serious damage and death to the host, particularly lemon, in Citrus and related genera, in Mediterranean countries and the Black Sea region. So far, however, it is unknown in Spain, Portugal and Morocco, as well as other major Citrus-growing regions of the world. Symptoms include red strands visible in stem xylem as well as veinal chlorosis, wilt and shedding of leaves, dieback of twigs and branches. Infection us through stomata and wounds. The causal organism, Phoma tracheiphila, is a conidial fungus. Once in the orchard, the fungus can be carried as spores from pycnidia and from hyphae on the plant and on fallen debris by rain, wind and irrigation water, and perhaps by birds and insects. Of concern to many international plant protection organizations, P. tracheiphila must be prevented from being spread in infected propagative material. Phoma tracheiphila (Petri) L.A. Kantsch. & Gikaschvili 1948 (Ascomycetes, Pleosporales) Mature pycnidia black, 60-165 x 45-140 µm, ostiolate, neck distinct, protruding into host epidermis, to 250 µm long. Conidia minute, unicellular, hyaline, 2-4 x 0.5-1.5 µm produced by phialides lining cavity, extruded through ostiole in whitish cirrhi. Single phialides 12-30 x 3-6 µm also borne on hyphae growing on exposed wood surfaces, wounded plant tissues and within host xylem elements. Conidia larger, hyaline, unicellular, straight or curved, with rounded apices, 3-8 x 1.5-3 µm. Blastoconidia (sensu Goidanich et al., 1948) ovoid to subpyriform, 15-17 x 7-9 µm, produced inside the xylem vessel of host and in agar culture. No teleomorph is known. For more information see; Petri (1930); Graniti (1955); Ciccarone (1971); Punithalingam and Holliday (1973) and Boerema et al. (1994). Distribution: Europe (Greece), Asia (Russia). Host: On stems of lemons and other Citrus spp. (Rutaceae). Notes: Originally described as Deuterophoma tracheiphila by Petri (1929, 1930), this fungus was transferred to the genus Phoma (Ciccarone and Russo, 1969). It is currently considered a member of the Phoma subgenus Plenodomus due to the presence of thick-walled cells of scleroplectenchyma tissue in the pycnidia (Boerema et al., 1994). Many species in this subgenus have Leptosphaeria teleomorphs and/or Phialophora synanamorphs (Boerema et al. 2004). Molecular phylogenetic studies have supported the status of P. tracheiphila in Phoma and shown a relationship to some Leptosphaeria species (Balmas et al. 2005). The production of conidia on "free" hyphae outside of pycnidia has been described as Acremonium-like (Petrie, 1929), as Cephalosporium to Cadophora-like (Goidanich and Ruggieri, 1947) and as a Phialophora sp. (Boerema et al., 2004). DISTRIBUTION Phoma tracheiphila occurs in most citrus-growing countries of the Mediterranean and Black Sea basins (CABI/EPPO, 2004), but has not been reported from Spain, Portugal or Morocco (Duran-Vila and Moreno, 2000; Licciardello et al., 2006; Migheli et al., 2009). In Turkey, it was restricted to a certain area when discovered in 1933, but spread later with the expansion of citrus plantations (Tuzcu et al., 1989). The presence of P. tracheiphila in Colombia, Uganda and Queensland, Australia has been reported, but these identifications are considered doubtful and the records are excluded from the distribution mapped by CABI/EPPO (2004). (See also Migheli et al., 2009). SIMILARITIES TO OTHER SPECIES The species of Phoma in the section Plenodomus, including P. tracheiphila, are difficult to identify in culture, because there is frequently little production of pycnidia and those produced do not have typical distinguishing morphology (Boerema et al., 2004). The limited host range of most species is useful; the species most morphologically similar to P. tracheiphila, P. coonsii, is found on apple (Malus pumila) in Japan and North America (Boerema et al., 2004). Species in the section Phoma that have a wide host range and may also appear on Citrus as saprophytes or secondary pathogens are difficult to identify in vitro (Boerema et al., 2004). The "mal secco" pathogen can be distinguished primarily by its production of conidia from phialides on "free" hyphae both on the plant and in culture (De Cicco et al., 1986; Boerema et al., 2004). A difficulty with isolation of Phoma tracheiphila is the frequent need to separate it from Colletotrichum gloeosporioides, which infects the lesions as a secondary pathogen but can grow faster in culture (Migheli et al., 2009). Colonies of the primary pathogen is easily distinguished from the reddish to orange colonies of the common epiphytic fungus, Epicoccum sp. (Migheli et al., 2009), whose clusters of dark, rough-walled, multicellular conidia (Ellis, 1971) are distinctive. The reddish pigments produced by P. tracheiphila on oatmeal agar after growth under alternating NUV light and darkness turn blue when concentrated sodium hydroxide (NaOH) is added (Boerema et al., 2004). In cultures on PDA, the red pigments form clusters of crystals on the mycelium and agar (Solel and Salerno, 2000). Other diseases that may result in sudden or slow wilting and dieback of citrus, sometimes with veinal chlorosis, are infection with the tristeza virus, if the tree has a sour orange rootstock, and Phytophthora root or foot rot, if the rootstock is virus-tolerant (Timmer et al., 2000). Huanglongbing, a systemic disease caused by an invasive bacterium in Asia and Africa, also causes slow decline of citrus trees, with yellowing of leaves and dieback of branches (Garnier and Bove, 2000). Drought, mechanical wounding, frost injury and other stresses can weaken trees and allow infections by weakly pathogenic fungi that result in branch cankers and dieback (Graham and Menge, 2000). DETECTION AND INSPECTION METHODS The leaves of affected trees should be checked for the chlorosis around veins that is an early symptom of the disease (Perrotta and Graniti, 1988). By stripping the bark or cutting into the wood, one can examine trees for the presence of the typical salmon-pink to orange-red discoloration of the xylem (Migheli et al., 2009). An ashy color of the dead twigs will result from lifting of the epidermis by growth of pycnidia produced underneath (Perrotta and Graniti, 1988). The fungus can be isolated from infected twigs on various solid media, including PDA (Magnano di San Lio and Perotta, 1986). DIAGNOSTIC METHOD Control of Phoma tracheiphila by exclusion, eradication, and treatment will be facilitated by rapid detection of the pathogen. Molecular techniques have been developed to provide a fast, specific and sensitive method that isolation in culture and morphological examination cannot. The standard PCR assay of Balmas et al. (2005) provides a more specific and sensitive test than the older one of Rollo et al. (1990); it distinguishes the pathogen from other Phoma species and detects its presence in both symptomatic and asymptomatic tissue, including wood. Another protocol was developed by Ezra et al. (2007) with specific concern for detection in fruit and distinguishing P. tracheiphila from other fungal pathogens of Citrus. Nevertheless, the standard method may not be sensitive enough for all monitoring or quarantine purposes (Demontis et al., 2007). Real-time PCR protocols have been published by Licciardello et al. (2006) and Demontis et al. (2007). These are capable of detecting pathogen DNA in picogram quantities in host tissue, so are suitable for quantitative work and detection of latent infections. Sequences of several regions of nuclear DNA, including those of the ITS region obtained by Balmas et al. (2005) are available for P. tracheiphila on GenBank (NCBI, 2010). NOTES ON CROPS AFFECTED Almost all Citrus species are susceptible to P. tracheiphila by inoculation (Perrotta and Graniti, 1988; Tuzcu et al., 1989). Lemon (C. limon) is probably the most affected crop (Perrotta and Graniti, 1988). Other susceptible species are citron (C. medica), bergamot (C. bergamia), mandarins (C. reticulata), and sour orange, (C. aurantium). In the field, hybrids of Citrus, related genera (Fortunella, Poncirus and Severinia), and other species have different degrees of resistance to the disease (Solel and Salemo, 2000). Most cultivars of sweet oranges ( C. sinensis), grapefruit (C. paradisi), and some mandarins and clementines ( Citrus deliciosa and C. reticulata), are only occasionally affected (Solel and Salemo, 2000). A number of rootstocks such as C. reshni, Poncirus trifoliata and, to a lesser extent, C. sinensis x P. trifoliata have been reported to be resistant (Perrotta and Graniti, 1988). Results of susceptibility tests can differ between investigators and between tests that use artificial inoculation and those that expose trees to natural infection (Tuzcu et al., 1989). SYMPTOMS The first symptoms appear in spring as shoot and interveinal leaf chlorosis followed by a dieback of twigs and branches. Raised black points within lead-grey or ash-grey areas of withered twigs indicate the presence of pycnidia. The growth of sprouts from the base of the affected branches and suckers from the rootstock are a very common response of the host to the disease. Individual branches or sectors may be infected initially (Solel and Salerno, 2000). Gradually the pathogen affects the entire tree, which eventually dies. When the wood of infected twigs, branches or trunks is cut or stripped of bark, the characteristic salmon-pink or orange-red discoloration of the wood may be seen; this internal symptom is associated with gum production within the xylem vessels (Perrotta and Graniti, 1988). In addition to the more common form of mal secco, two other forms of the disease are distinguished: "mal fulminante" in which the tree declines and dies rapidly, apparently due to root infection, and "mal nero" which is a chronic infection of the tree that causes a browning of the heartwood (Perrotta and Graniti, 1988). BIOLOGY AND ECOLOGY BIOLOGY AND ECOLOGY Life cycle: The fungus enters through wounds in leaves, branches and roots; penetration through stomata is questionable (Zucker and Catara, 1985; Perrotta and Graniti, 1988). Cultivation practices as well as wind, frost and hail, that cause injuries to various parts of the trees favor infection. Inoculum may be provided both by conidia produced in pycnidia on withered twigs and by conidia produced from phialides borne on hyphae on exposed woody surfaces of the tree or on debris on the ground. The conidia are thought to be waterborne for the most part (Solel, 1976). The range of temperature at which infection will occur is between 14 and 28°C. In the Mediterranean region, infection periods depend on local climatic and seasonal conditions; in Sicily, infections usually occur between September and April (Somma and Scarito, 1986). The length of the incubation period may vary according to the season (Grasso and Tirrò, 1984). The optimum temperature for growth of the pathogen and symptom expression is 20-25°C, whereas the maximum temperature for mycelial growth in culture is 30°C. (Perrotta and Graniti, 1988). Discarded prunings containing affected twigs or branches can be a source of inoculum for several weeks. A relative humidity near saturation and temperatures between 20 and 25°C are best for production of phialides on hyphae in wounds and natural leaf scars (De Cicco et al., 1986). The fungus can survive within infected twigs in the soil for more than four months (De Cicco et al., 1987). Reproductive biology: No teleomorph is known, and the genetic uniformity among isolates in Italy (Balmas et al., 2005) and Israel (Ezra et al., 2007) indicates that sexual recombination is not commonly occurring in these Mediterranean regions. Physiology and phenology: Phoma tracheiphila produces an extracellular glycoprotein toxin that reproduces the symptoms of veinal chlorosis, necrosis and wilt when injected into lemon leaves (Nachmias et al., 1977). This toxin has been named "malseccin" (Reverberi et al., 2008) In Italy, two types of strains have been reported: chromogenic strains that produce a red pigment in culture, and non-chromogenic strains that do not produce this pigment but show a yellow color (Graniti, 1969). Limited data indicate no difference in pathogenicity that is clearly related to this variation, although variation in virulence does occur in P. tracheiphila (Magnano di San Lio et al., 1992; Magnano di San Lio and Perrotta, 1986). Among 600 isolates from lemon in Sicily, all but a few were chromogenic , and there were no remarkable differerences in colony morphology within either type. On some media, some of the chromogenic isolates underwent sectoring to yield a variant that does not produce pycnidia (Magnano di San Lio and Perrotta, 1986). In Israel, both chromogenic and non-chromogenic strains could be isolated from a single tree (Ezra et al., 2007). Associations: Acervuli of the Colletotrichum state of Glomerella cingulata, a secondary invader of withered twigs, are often associated with the pycnidia of P. tracheiphila (Migheli et al., 2009). MOVEMENT AND DISPERSAL Natural dispersal: Conidia are dispersed from the trees and debris by rain splash or overhead irrigation (Solel and Salerno, 2000). Some may become airborne; Balmas et al. (2005) obtained isolates in Italy from air samples. Laviola and Scarito (1989) found the fungus spreading only a short distance, between 15 and 20 m, from a source of inoculum, although the prevailing wind direction did increase the distance. Vector transmission: Transmission by vectors has not been reported, but is possible. Birds and insects have been suggested as non-specific carriers of the fungus between trees (Perrotta and Graniti, 1988). Accidental introduction: Systemic infection due to release of spores in the xylem will result in most parts of the tree being able to carry the fungus. The pathogen has been detected in asymptomatic parts (Balmas et al., 2005; Ezra et al., 2007). Isolation from lemon seed was reported (Stepanov and Shaluishkina, 1952) but seedborne transmission to new trees was not demonstrated. If not done carefully, pruning, whether or not done for disease control, may contaminate the tools used. SEEDBORNE ASPECTS OF DISEASE Incidence Phoma tracheiphila has been detected in lemon seeds (Stepanov and Shaluishkina, 1952). There is no evidence that it is seedborne in other species. IMPACTS Economic impact: In the Mediterranean region, P. tracheiphila is the most destructive fungal pathogen of lemons. Up to 100% of trees in an orchard of a susceptible lemon cultivar can be affected (Perrotta and Graniti, 1988). Destructive outbreaks of the disease may occur after frost spells and hail storms in spring (Perrotta and Graniti, 1988). In general, injury to the tree through severe cold weather may predispose it to fungal attack. The symptoms of the disease are most severe in spring and autumn; at high summer temperatures, spread of the fungus in the vascular system ceases and the symptoms do not develop further (Ruggieri, 1953). In the areas where the pathogen is present, the disease reduces the quantity and quality of lemon production and limits the use of susceptible species and cultivars. It has been estimated that complete control of the pathogen could cause a doubling of lemon harvests (Gulsen et al., 2007). The potential impact of invasion by this fungus on the lemon industry alone may be estimated from the fact that fresh lemon fruit are produced and shipped from countries on five continents (Migheli et al., 2009). Restrictions to prevent importation of possibly infected fruit of additional Citrus species could create economic damage in other countries. MANAGEMENT SPS measures (quarantine, certification, prohibition) In countries or areas where the disease has not yet been reported, great care must be taken not to use plants from nurseries located in areas where the pathogen occurs for new plantings or replantings. Phoma tracheiphila is on the A2 list of quarantine organisms for the European and Mediterranean Plant Protection Organization for whom the threat is most immediate. It is of concern for exclusion by other regional plant protection organizations worldwide (Migheli et al, 2009). Eradication In 1998, the government of Italy mandated the destruction of infected plant material, including the uprooting and burning of the tree stumps (Migheli et al., 2009). Cultural control and sanitary measures Phoma tracheiphila may be kept under control by pruning infected twigs as soon as the first symptoms appear (Salerno and Cutuli, 1982). Common practices aimed either at saving the affected trees or at eradicating the inoculum include removal of whole branches and grafting the resulting pollarded tree with resistant cultivars or species. Burning of the pruned branches is recommended in order to eliminate possible sources of inoculum (Solel and Salerno, 2000). Injury during cultural practices must be avoided; cultivation of the orchard in the fall may create wounds in the roots that will be slow to heal (Duran-Vila and Moreno, 2000). BIOLOGICAL CONTROL Infection with the citrus exocortis viroid was shown to prevent infection by the fungus (Solel et al., 1995). Trees have been inoculated with a hypovirulent strain of the pathogen in an effort to achieve a similar effect (Solel and Salerno, 2000). Cirvilleri et al. (2005) found a genetically heterogeneous group of rhizosphere-inhabiting pseudomonad bacteria, mostly Pseudomonas fluorescens and P. putida, that inhibited Phoma tracheiphila growth in culture; 248 isolates were obtained from both wild-type and transgenic rolABC rough-lemon rootstocks and 13 from only the genetically modified plants. The possible role these organisms could have in Citrus protection was not examined. CHEMICAL CONTROL Chemical control is not widely used except in nurseries. Copper fungicides and ziram are the most common products used. The protectant copper fungicides will need to be applied repeatedly to the canopy during the period of greatest susceptibility from autumn to spring (Solel and Salerno, 2000). Systemic products such as carboxin and benzimidazole are also effective only as preventives (Solel and Salerno, 2000) and some that were effective as soil drenches in pot tests were found ineffective in the field (Solel et al., 1972). A mixture of a protectant and a systemic fungicide is recommended by Duran-Vila and Moreno (2000), particularly after weather conditions, such as a freeze, hailstorm, or strong winds that cause wounds to the tree. HOST RESISTANCE The best method of controlling the disease would be to grow resistant clones, but unfortunately this is not easily achieved. In some areas of Sicily, the susceptible cultivar Femminello has been replaced by the resistant lemon cultivar Monachello, which is of inferior quality. Resistant clones of cv. Femminello have been selected, but they either do not adapt to different environmental conditions or have still to be tested in the field (Gentile et al., 1992; Salerno and Cutuli, 1992). The cultivar Santa Teresa was recommended as resistant and suitable for Greece (Protopapadakis and Zambettakis, 1981). Recently, in Turkey, selection and breeding have resulted in a good genotype of lemon, Tuzcu 894, which has high yield per tree, good fruit weight, high juice content and low seed number, and so may be a replacement for the tree, good fruit weight, high juice content and low seed number, and so may be a replacement for the common agronomically desirable but susceptible cultivar, Kutdiken (Uzun et al., 2009). Various more rapid modern means have been used to try to obtain resistant germplasm. Somatic hybrids of susceptible and resistant lines were of intermediate resistance (Tusa et al, 2000). Budwood was irradiated to produce mutant tissue; types with varied levels of resistance were obtained (Gulsen et al., 2007). Protoplasts (Gentile et al., 1992) and culture callus (Bas and Koc, 2006) of Citrus species have been exposed to the toxin malseccin as a means of selection. Most recently, Femminello lemon plants have been transformed with a Trichoderma harzianum endo-chitinase gene coding for a protein that is then produced in the leaves and inhibits growth of the pathogen in culture (Gentile et al., 2007). GAPS IN KNOWLEDGE/RESEARCH NEEDS The reasons why the pathogen is not a problem in countries at the western end of the Mediterranean region may have significance for control of the disease. Exploration of possible biological controls, acting either outside or inside the tree, should be continued. References

Suggested citation: Chalkley, D..Systematic Mycology and Microbiology Laboratory, ARS, USDA. . Invasive Fungi. Mal secco disease of Citrus - Phoma tracheiphila . Retrieved September 25, 2021, from /sbmlweb/fungi/index.cfm . Use this link to revisit SMML website Trees killed due to mal secco disease. Photo courtesy of 2. Living tree showing symptoms of mal secco disease. CABI Crop Protection Compendium. Photo courtesy of CABI Crop Protection Compendium.

Wood stained by Phoma tracheiphila. Photo courtesy of CABI Crop Protection Compendium.