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Chapter 1
Major and Emerging Fungal Diseases of Citrus in the
Mediterranean Region
Khaled Khanchouch, Antonella Pane, Ali Chriki and
Santa Olga Cacciola
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/66943
Provisional chapter
© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
Major and Emerging Fungal Diseases of Citrus in the
Mediterranean Region
Khaled Khanchouch, Antonella Pane, Ali
Chriki and Santa Olga Cacciola
Additional information is available at the end of the chapter
Abstract
This chapter deals with major endemic and emerging fungal diseases of citrus as well as
with exotic fungal pathogens potentially harmful for citrus industry in the Mediterranean
region, with particular emphasis on diseases reported in Italy and Maghreb countries.
The aim is to provide an update of both the taxonomy of the causal agents and their
ecology based on a molecular approach, as a preliminary step towards developing or
upgrading integrated and sustainable disease management strategies. Potential or actual
problems related to the intensication of new plantings, introduction of new citrus cul-
tivars and substitution of sour orange with other rootstocks, globalization of commerce
and climate changes are discussed. Fungal pathogens causing vascular, foliar, fruit, trunk
and root diseases in commercial citrus orchards are reported, including Plenodomus tra-
cheiphilus, Colletotrichum spp., Alternaria spp., Mycosphaerellaceae, Botryosphaeriaceae,
Guignardia citricarpa and lignicolous basidiomycetes. Diseases caused by Phytophthora
spp. (oomycetes) are also included as these pathogens have many biological, ecological
and epidemiological features in common with the true fungi (eumycetes).
Keywords: Plenodomus tracheiphilus, Colletotrichum spp., Alternaria spp., greasy spot,
Mycosphaerellaceae, Botryosphaeriaceae, Guignardia citricarpa, Basidiomycetes,
Phytophthora spp
1. Introduction
Citrus are among the ten most important crops in terms of total fruit yield worldwide and
rank rst in international fruit trade in terms of value. More than seven million hectares are
planted with citrus throughout the world. The term “citrus” indicates a complex of species
and hybrids of the genera Citrus, Eremocitrus, Fortunella, Microcitrus and Poncirus, subfamily
© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Aurantioideae (family Rutaceae). It is assumed that all presently cultivated citrus species origi-
nate from three ancestral “true” species, Citrus medica (citron), Citrus reticulata (mandarin) and
Citrus maxima (pummelo). Although citrus are native to East Asia, citriculture has expanded
in tropical, subtropical and Mediterranean climatic regions, and presently Mediterranean
countries are the leading producers for the international fresh market. The term “fungal”
diseases referred to citrus pathologies includes both diseases caused by “true” fungi or fungi
sensu stricto (eumycota) and those caused by oomycetes. In fact, although Oomycetes are
part of a distinct kingdom (Chromista or Stramenopiles), they are traditionally considered
fungi sensu lato as they have in common with Eumycota some ecological and morphological
(e.g. lamentous hyphae) features. This chapter is not intended to be a complete review of
fungal diseases reported in the Mediterranean citrus belt. It deals only with major endemic
and emerging fungal diseases of citrus reported in Italy and Maghreb countries, as well as
with exotic fungal pathogens potentially harmful for citrus industry in the Mediterranean
region. The aim is to provide an update of the taxonomy of the causal agents and their ecol-
ogy and diagnosis based on a molecular approach as a preliminary step towards developing
or upgrading integrated and sustainable management strategies. Postharvest fungal diseases
of citrus are not within the scope of this brief review.
2. General considerations
Any rational disease management strategy is based on accurate diagnosis and prevention.
Fungal diseases of citrus showing specic symptoms can be easily diagnosed visually, while
for diseases with no typical symptoms, laboratory tests are needed. A limit of symptomatic
diagnosis is that some symptoms are visible only at certain times of the year or appear on
organs distinct from those colonized by the pathogen. Moreover, secondary parasites or
opportunistic pathogens can overgrow primary pathogens or colonize senescent or necrotic
tissues. Typical examples are Colletotrichum species such as the cosmopolitan Colletotrichum
gloeosporioides, the most common Colletotrichum species on citrus and Colletotrichum karstii. C.
karstii has recently been described as a separate species of the Colletotrichum boninense com-
plex, using a multilocus molecular phylogenetic analysis [1]. It is a widespread and polypha-
gous species; however, so far it has been reported on citrus only in Italy and China [2, 3], very
probably reecting a sampling bias. Colletotrichum species are associated with citrus as endo-
phytes, saprobes as well as pre- and postharvest anthracnose pathogens. They can switch
their lifestyle from endophytes to saprobes or opportunistic pathogens and produce acervuli
on necrotic tissues as a consequence of biotic and abiotic stresses, such as mal secco disease,
frost, wind, hail and any type of mechanical injury.
The most eective control method of fungal diseases is prevention, especially for diseases
caused by soil-borne pathogens. Most of the rootstocks used in commercial citrus orchards,
e.g. are resistant to Phytophthora trunk gummosis and root rot. Genetic resistance has also
been used in citriculture for prevention of vascular and canopy diseases such as mal secco
disease of lemon and brown spot of tangerines, respectively. However, the choice of the scion
is primarily conditioned by commercial requirements. The genetic susceptibility to fungal
Citrus Pathology4
diseases is a limiting factor to the widespread diusion of some citrus cultivars [‘Fortune’
mandarin, e.g. is not planted in humid areas as it is very susceptible to Alternaria brown spot
(ABS)]. Prevention methods include selection of the plantation site, surface levelling of the
ground to avoid waterlogging, soil drainage and rational management of irrigation. Irrigation
systems that wet the trunk favour Phytophthora gummosis, but risk is reduced if trees are
irrigated during the morning in order to allow the bark to dry quickly. Sprinkler irrigation
under the canopy is conducive to Phytophthora infections on trunk and fruits, while overhead
sprinkler irrigation favours brown spot epidemics in orchards of susceptible tangerine culti-
vars as well as Septoria spot and mal secco in lemon orchards. Generally speaking, localized
irrigation methods, such as drippers, are less conducive to leaf and fruit diseases. Usually,
fungal infections occur on citrus trees irrespective of their vigour. However some diseases
aack only weakened trees, while others develop preferentially on vigorous trees. Alternaria
brown spot, e.g. is more severe on trees with dense canopy and copious spring vegetation as
the causal agent, Alternaria alternata (A. alternata), sporulates only on young leaves and with
high relative humidity. As a consequence this disease is favoured by intensive plantings and
high amounts of nitrogen fertilizers.
Mediterranean climate is not conducive to epidemic infections of fungal diseases of the tree
canopy, and as a consequence, chemical control of these diseases in the Mediterranean region
is economically justied only in few cases. The choice of fungicides is restricted to active
ingredients registered for citrus. In Italy, only copper derivatives (oxychloride, hydroxide
and sulphate tribasic) are allowed for eld treatments against fungal diseases. Systemic fun-
gicides, metalaxyl M and ethyl-phosphytes (Al ethyl-phosphite and K ethyl-phosphite) are
available for chemical control of diseases caused by Phytophthora. Metalaxyl M can be applied
as soil drench or trunk paint as it is translocated through the plant apoplast. The derivates of
phosphorous acid are translocated through the symplast so they can be applied to the trunk
(as paints or sprays) or the tree canopy (as leaf sprays). In some commercial products, cupric
and systemic fungicides are blended together.
Hereafter we illustrate major fungal diseases of citrus already established or potentially
harmful for the citrus industry in the Mediterranean region. Two quarantine fungal patho-
gens of citrus presently are included in the A1 list of the European and Mediterranean Plant
Protection Organization (EPPO), Guignardia citricarpa (G. citricarpa) (anamorph, Phyllosticta
citricarpa), the causal agent of fruit black spot, and Pseudocercospora (Phaeoramularia) angolensis
(P. angolensis), the causal agent of angular leaf spot also known as Phaeoramularia leaf and
fruit spot. However, only for G. citricarpa an ocial diagnostic protocol is available. The EPPO
A1 list includes pathogens and parasites whose introduction in the territory of EPPO would
cause severe phytosanitary risks. Import of citrus fruits and propagative material from areas
where these pathogens are present is subject to customs restrictions. Geographic distribution
of G. citricarpa includes Asia, Africa, Australia, South America and Florida. A real risk for the
citrus industry in Italy is that this fungal pathogen can be imported with citrus from South
Africa to South America. In the last years, this pathogen has been intercepted several times
on citrus imported from South Africa, Brazil and Uruguay. For more details on citrus black
spot, refer to the EPPO diagnostic protocol PM7/17 (hp://www.eppo.int) [4]. The protocol
reports ocial molecular diagnostic methods to identify the causal agent of black spot and
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
http://dx.doi.org/10.5772/66943
5
distinguish it from the non-pathogenic species Guignardia mangiferae. P. angolensis causes leaf
spots and necrotic lesions on fruits. Geographic distribution of this pathogen is restricted to
the warm and humid areas of central Africa, at altitudes between 80 and 1500 m, and Yemen,
in Asia. All species of cultivated Citrus appear to be susceptible.
3. Mal secco
The mal secco, an Italian name meaning “dry disease”, is a vascular wilt disease (Figure 1) caused
by the mitosporic fungus Plenodomus tracheiphilus (P. tracheiphilus), formerly Phoma tracheiphila.
Symptoms include strands of salmon-pink to orange-red discoloration visible in stem xylem
(Figure 2) as well as veinal chlorosis (Figure 3), wilt and shedding of leaves, dieback of twigs
and branches. The disease is particularly destructive on lemon (Citrus limon) in Mediterranean
countries and the Black Sea region. So far, however, it has not been reported in Spain, Portugal
and Morocco, as well as other major citrus-growing regions of the world, even though there is
no obvious climatic or cultural factor limiting its establishment in uninfested areas.
Figure 1. Wilting, leaf shedding and defoliation on a young lemon tree aected by mal secco (courtesy S.O. Cacciola, G.
Magnano di San Lio, A. Pane).
Citrus Pathology6
Figure 2. Pink-salmon discoloration of the wood associated to mal secco disease (courtesy S.O. Cacciola and A. Pane).
Figure 3. Clearing and chlorosis of a sour orange leaf aected by Plenodomus tracheiphilus (courtesy S.O. Cacciola and
A. Pane).
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
http://dx.doi.org/10.5772/66943
7
P. tracheiphilus is a quarantine pathogen of great concern to many international plant pro-
tection organizations; it is listed on the list A2 of EPPO and the lists of quarantine patho-
gens of Asia and Pacic Plant Protection Commission (APPPC), Caribbean Plant Protection
Commission (CPPC), Comité Regional de Sanidad Vegetal del Cono Sur (COSAVE), North
American Plant Protection Organization (NAPPO) and Inter-African Phytosanitary Council
(IAPSC). Moreover P. tracheiphilus was included in a list of microorganisms that have to be
regarded as potential biological weapons as they cause destructive diseases of economically
relevant crops [5]. Although lemon is the principal host, other species of citrus and related
genera (Fortunella, Poncirus and Severinia) may be infected [6]. In several Mediterranean
countries, including Greece, Israel, Italy and Tunisia, severe infections have been sporadi-
cally observed, also in commercial orchards of bergamot, citron, sweet oranges, tangerines,
mandarins and mandarin hybrids. Many of these reports on sweet oranges, tangerines,
mandarins and mandarin hybrids refer to the chronic facies of the disease known as “mal
nero” (Figure 4), very probably originating in the nursery. Young seedlings of common cit-
rus rootstocks such as sour orange (Citrus aurantium), citranges (Citrus sinensis ‘Washington’
sweet orange × Poncirus trifoliata) and citrumelo ‘Swingle’ (Citrus paradisi ‘Duncan’ grape-
fruit × P. trifoliata) proved to be susceptible to natural infections in nursery.
Figure 4. The ‘mal nero’ facies of mal secco disease (courtesy S.O. Cacciola, G. Magnano di San Lio, A. Pane).
Citrus Pathology8
Alemow (Citrus macrophylla) is extremely susceptible, and trees on alemow rootstock may die
as a consequence of root infections. This facies of the disease associated to root infections is
known as “mal fulminante”, an Italian name meaning “sudden death”.
The mal secco fungus was originally classied among the Deuteromycota as Deuterophoma tra-
cheiphila. Later, it was reclassied as P. tracheiphila and considered a member of the subgenus
Plenodomus due to the presence of thick-walled cells in the pycnidial scleroplectenchymatous
tissues [7]. Many species in this subgenus have Leptosphaeria teleomorphs and/or Phialophora
synanamorphs [8]. Although a molecular phylogenetic study has shown a relationship
between P. tracheiphila and Leptosphaeria species [9], the teleomorph of the mal secco fungus
has not yet been identied.
Recent molecular phylogenetic studies on the genus Phoma demonstrated its polyphyly and
lead to the denition of the genus Plenodomus. P. tracheiphila and many other fungi of the sec-
tion Plenodomus were allocated within this genus [10–12]. Presently, therefore the accepted sci-
entic name of the causal agent of mal secco disease of citrus is P. tracheiphilus. Identication
of P. tracheiphilus is currently based on morphological and molecular methods as described in
the OEPP/EPPO diagnostic protocol PM7/048(3) (hp://www.eppo.int) [13]. In the last years,
numerous molecular techniques based on conventional polymerase chain reaction (PCR) and
real-time PCR (rtPCR) have been developed, allowing fast and sensitive detection and quan-
tication of the pathogen from infected tissues [9, 14–17]. Some of these techniques have been
applied in breeding programmes to monitor the progress of the fungus in the host xylem and
test the susceptibility of lemon cultivars to mal secco. As in other tracheomycoses, in fact,
there is a direct correlation between the rate and extent of xylem colonization by the pathogen
(i.e. the amount of the fungus in the vessels) and the susceptibility of the cultivar to the dis-
ease as determined by symptom severity.
P. tracheiphilus can infect citrus hosts all year round, but most infections occur from September
to April. Pycnidiospores produced within pycnidia and phialoconidia produced on mycelium
are the infective propagules. Pycnidia are present throughout the year on withered twigs
(Figure 5) and branches; pycnidiospores are extruded when the weather is wet. Phialoconidia
are produced on the surface of infected tissues exposed by wounds or on woody debris in
soil. Penetration occurs through wounds. Penetration through natural opening has been
hypothesized but never demonstrated. After penetration, the mycelium grows in the lumen
of xylem vessels producing both phialoconidia and blastoconidia that are translocated by
the ascendant lymph and invade the wood. The pathogen can be detected distantly from
its penetration site, by molecular tools, before the expression of the disease symptoms [17].
Systemic invasion by the pathogen leads to the loss of xylem functionality and the appearance
of water stress symptoms typical of tracheomycoses. Impairment of water transport is proved
by the increase of hydraulic resistance of the stem and leaves as well as the closure of stomata
[18, 19]. Although the optimum temperature for pathogen growth is about 25°C, optimum
temperature for symptom expression and xylem colonization is 20–22°C.
Infection occurs between 14 and 28°C, whereas at temperatures above 28°C, fungal growth
ceases and symptoms are not expressed. As a consequence disease progress is temporarily
inhibited during the hot or cold temperature extremes.
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
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9
While in Sicily most infections occur from autumn to early spring; in Israel, mid-November
to mid-April was the most conducive time for infection, coinciding with the rainy season,
although no correlation was found between the amount of rain, the number of rainy days
and the percentage of infected plants. No infection was observed after the rain ceased, so it
appears that the rain aects inoculum dissemination rather than infection [20]. Length of the
incubation period varies according to season, and in young trees, it ranges between 2 and 7
months, whereas it can last several years in the “mal nero” form of the disease because this
chronic infection could remain conned to the heartwood over a long time. Expression of
symptoms is therefore a poor selection criterion for phytosanitary inspection of propagation
material. This aspect has practical relevance as the use of disease-free propagation material
helps to reduce the dissemination of mal secco and its introduction into disease-free areas.
Like for other tracheomycoses, fungicide treatments are not eective against mal secco, and
research of newly resistant genotypes remains the only eective strategy to control this dis-
ease. Lemon cultivars with various degrees of resistance to mal secco, such as ‘Monachello’,
‘Interdonato’, ‘Feminello Zagara Bianca’, ‘Femminello Continella’ and ‘Cerza’, have been selected
in Sicily. However, ‘Monachello’ has a poor yield, ‘Interdonato’ does not bloom several times,
Figure 5. White-red 1–2-year-old twig of lemon with pycnidia of Plenodomus tracheiphilus. Pycnidia appear as scaered
black spots on the dried portion of the twig (courtesy S.O. Cacciola and A. Pane).
Citrus Pathology10
and its juice has low acidity, and the tolerance to mal secco of the other cultivars is not compara-
ble to that of ‘Monachello’. Two new cultivars with high yield potential, ‘Femminello Siracusano
2Kr’, a mutant nucellar clone obtained with cobalt γ-radiation, and the triploid hybrid ‘Lemox’
(European patent number 20040073), have been included in the ocial list of lemon cultivars
whose use is recommended in Italy for new plantings. However they proved to be extremely
susceptible to mal secco disease. The goal of obtaining tolerant cultivars with competitive yields
and satisfactory bio-agronomic characteristics remains one of the primary objectives of lemon-
breeding programmes in the Mediterranean region. Additional and more detailed information
on mal secco disease can be found in two recent comprehensive reviews [20, 21].
4. Emerging and endemic foliar and fruit diseases
Despite the Mediterranean climate is not conducive to epidemic outbreaks of fungal diseases
of the canopy of citrus trees, being characterized by long periods of drought and high tem-
peratures in summer as well as cool winters, in the last years, some citrus-growing areas of
the Mediterranean region have experienced the emergence or resurgence of new and endemic
fungal diseases of leaves and fruit.
4.1. Alternaria brown spot
Alternaria brown spot (ABS) is one of the most important diseases of tangerines and their hybrids
worldwide. It is caused by the tangerine pathotype of the fungus A. alternata [22, 23]. A. alternata is
a typical necrotrophic pathogen. It produces a host-specic (hs) toxin named ACT-toxin (ACTT),
which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in suscepti-
ble citrus genotypes. There are several pathotypes of A. alternata characterized by host specicity
[24]. The chemical structure of ACT-toxin is similar to those of other hs-toxins such as AK- and
AF-toxin, produced by the Japanese pear and strawberry pathotypes, respectively [25, 26]. The
tangerine pathotype of A. alternata carries a gene cluster (ACTT) located in a small chromosome
which is responsible for the biosynthesis of ACT-toxin [27]. There is also indirect evidence sug-
gesting the presence of toxin receptors in susceptible citrus genotypes. In addition, recent studies
conrm that the ACT-toxin is a pathogenicity factor and indicate that the mitigation of reactive
oxygen species (ROS) produced by the host plant is essential for the pathogenicity of A. alter-
nata. A signicant correlation was found between pathogenicity on citrus leaves and ACTT gene
expression in isolates of A. alternata from citrus of various geographic origins [28]. ACT-toxin is
released during the germination of conidia. It induces necrotic areas of the leaf blade and may be
translocated to the vascular system inducing chlorosis and necrosis along the veins.
ABS is prevalent in citrus production areas with a Mediterranean climate, characterized by
cool, humid winters and hot, arid summers. It was rst reported on ‘Emperor’ mandarin in
Australia in 1903, and subsequently it was detected in the Americas, the Mediterranean basin,
South Africa, Iran and China aecting mainly ‘Fortune’ and ‘Nova’ mandarin hybrids [22, 29,
30]. In Europe, it has been reported in Greece, Italy and Spain. Its appearance in Italy coin-
cided with the diusion of the mandarin ‘Fortune’. Warm temperatures and prolonged wet-
ness are required for infection. However the disease causes severe epidemics in both humid
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
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11
areas and semi-arid regions provided a susceptible citrus variety is present. Fruits can get
infected in all development stages but are more susceptible during the rst four months fol-
lowing petal fall. Spring infections on young fruits may lead to premature fruit drop. Early
fruit drop is common, especially if infection has occurred shortly after petal fall. Symptoms
on fruits are necrotic brown circular lesions that may vary in size (Figures 6 and 7). Mature
Figure 7. Alternaria brown spot on ‘Nova’ tangelo hybrid (courtesy S.O. Cacciola, G. Magnano di San Lio, A. Pane).
Figure 6. Typical symptoms of Alternaria brown spot on ‘Fortune’ mandarin (courtesy S.O. Cacciola, G. Magnano di
San Lio, A. Pane).
Citrus Pathology12
lesions have a corky appearance, and in older lesions, the centre may dislodge leaving tan-
coloured pockmarks. Brown to black lesions surrounded by yellow halos and veinal necrosis
appear on young leaves, which often are deformed due to necrosis of the margin. On highly
susceptible cultivars abundant defoliation, abscission of young shoots and twig dieback may
occur. Conditions of persistent humidity (fog or dew), which provide a weing period of 8–12
h, are conducive for the development of infections; the optimum temperature is 20–27°C, but
infections can occur between 17 and 32°C. The disease incubation period is 16–36 h. Conidia
are produced on necrotic lesions in young leaves but not on fruits and are dispersed by air
currents and rain splash. The presence of the disease is a limiting factor for the diusion of
highly susceptible mandarin or tangerine-like cultivars such as ‘Fortune’, ‘Dancy’, ‘Minneola’,
‘Orlando’, ‘Nova’, ‘Guillermina’, ‘Clemenpons’, ‘Esbal’, ‘Page’, ‘Lee’, ‘Sunburst’, ‘Encore’,
‘Murco’, ‘Michal’, ‘Winola’, ‘Ponkan’, ‘Emperor’, ‘Tangfang’ and ‘Primosole’. Even some vari-
eties of pomelo are susceptible, while orange cultivars, with very few exceptions, are resistant.
Lemon and lime cultivars are not susceptible, with the exception of Mexican lime (Citrus
aurantifolia) which is slightly susceptible.
Generally speaking, hybrids with ‘Dancy’ and ‘King’ mandarins as a parent are very suscep-
tible. In many countries, such as Italy, Israel, Spain and the USA, ABS is a strong concern for
triploid breeding programmes aiming at producing seedless mandarin cultivars. From dip-
loid progeny analysis, it has been proposed that the inheritance of ABS susceptibility in citrus
is controlled by a single gene with two alleles, one dominant (S) and the other recessive (r)
which transmit susceptibility and resistance, respectively [31]. Therefore, resistant cultivars
are considered to be recessive homozygous for this locus, whereas susceptible cultivars could
be heterozygous or homozygous dominant. Cultivars like ‘Minneola’ and ‘Dancy’, which are
homozygous (SS), transmit susceptibility to all the descendants. Most susceptible commercial
cultivars like ‘Fortune’, ‘Nova’ and ‘Murco’ are heterozygous, and both resistant and sus-
ceptible hybrids can be found in their progeny.
Resistant oranges, mandarins and clementines are recessive homozygous (rr), so when they
breed with each other, all descendants are resistant. The single-locus dominant inheritance of
susceptibility was corroborated by the analysis of triploid progenies. Recently, in Spain two
new ABS-resistant hybrids of ‘Fortune’, ‘Garbi’ (‘Murco’ × ‘Fortune’) and ‘Safor’ (‘Kara’ ×
‘Fortune’) have been released.
Currently on susceptible cultivars, ABS control is based on the application of fungicides [32,
33]. Sprays must be scheduled to protect susceptible organs during the critical period for
infection. Depending on the climate and the susceptibility of the cultivar, between four and
ten fungicide sprays per year may be needed to produce quality fruit for the fresh market.
On susceptible cultivars, foliar applications with copper fungicides are requested every 10–15
days in periods of high susceptibility. Despite this large number of sprays, disease control
is not always satisfactory, and cultivation of very susceptible cultivars such as ‘Fortune’ in
Mediterranean countries and ‘Minneola’ in Florida has declined signicantly.
An integrated approach can reduce the risk of ABS infections and the disease severity [22].
In the nursery, it is recommended to grow susceptible citrus cultivars indoors, to avoid
infections on young shoots and prevent inoculum dissemination in new commercial citrus
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
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plantings. New plantings of susceptible cultivars should be established in ventilated sites
where environmental conditions are unfavourable for infections and sporulation of the causal
agent on young leaves. Similarly, dense planting is not recommended for susceptible culti-
vars. Orchards of susceptible cultivars should be monitored frequently to detect the presence
and prevent epidemic outbreaks of the disease.
4.2. Septoria spot
Septoria is a genus of plant pathogenic fungi with a wide geographic distribution, commonly
associated with leaf spots and stem cankers of a broad range of host plants. Species of Septoria
are among the most common and widespread leaf-spoing fungi worldwide. The causal agent
of Septoria spot of citrus has been identied as Septoria citri [34]. This disease has been found
in many citrus-producing countries of the Mediterranean basin, South Africa, South America,
Australia (including Tasmania, Eastern and Southern Australia), India and California. Lemon
(C. limon) and grapefruit (C. paradisi) are the most frequently damaged Citrus species world-
wide, but all commercial citrus cultivars are susceptible. In Australia grapefruit, lemon and
sweet orange (C. sinensis) ‘Washington navel’ are regarded as the most susceptible hosts.
‘Valencia’ oranges for juice production can also be aected although this cultivar is consid-
ered less susceptible than ‘Washington Navel’. In California, Septoria spot aects ‘Valencia’
oranges, late-season navel oranges and occasionally lemons and grapefruits. It occurs in the
San Joaquin Valley and interior districts of southern California during cool, moist weather.
In Italy, Septoria spot has been reported on lemon, clementine and bergamot. Surprisingly, S.
citri is a quarantine organism for Western Australia and South Korea. In 2004, Korean National
Plant protection and Quarantine Service (NPQS) detected and rejected citrus fruits infected
with Septoria spot imported from California. In most citrus-producing countries, Septoria spot
is generally considered a disease of minor signicance, except for fruit produced for the fresh
market as rind blemishes reduce fruit quality aesthetically and aect saleability. Symptoms
on fruit are small (1–2 mm in diameter), round, light tan-coloured lesions (pits) with a nar-
row green margin on the outer rind. As the fruit matures, they become reddish to pale brown
(Figure 8) and contain small black spots (S. citri pycnidia) barely visible to the naked eye.
When frost occurs or during storage fruit, lesions may enlarge (3–10 mm in diameter) and
merge to form brown-to-black sunken blotches. These may be several centimetres in diam-
eter and extend to the inner rind (albedo) and occasionally into the fruit segments. In severe
infections, fruits develop an o avour and drop prematurely. Symptoms may not appear
until fruit is in storage. Leaf symptoms incited by S. citri are initially conned to the lower
surface of the leaf and consist of small, blister-like brown to black spots, 1–4 mm in diameter,
surrounded by a yellow halo. After leaf fall the spots turn brown with a dark margin and the
pycnidia of the fungus form on necrotic tissues. Where under-canopy irrigation is used, infec-
tion may result in severe defoliation of the lower part of the tree (canopy skirt).
Septoria spot is more severe in years when rainfall levels are high and temperature uctuates.
The causal agent survives in infected orchards as a saprobe. Inoculum is constituted by pyc-
nidia forming on dead twigs and leaves. Conidia, the infective propagules, are dispersed by
water splash. Infections usually occur during cool, damp weather in late summer or autumn
and when the fruits are still green. They may remain latent for up to six months, and fruit
symptoms generally appear as the fruit starts ripening in late winter and early spring, after
Citrus Pathology14
cool, frosty or cold windy weather. The susceptibility of fruits is related to the maturity of the
rind at the time of infection. Management practices to prevent or reduce the disease incidence
and severity include tree skirting and canopy pruning to improve air circulation, early fruit
harvesting and the removal of withered twigs from the tree canopy and fallen leaves from
the soil under the tree canopy to reduce the amount of inoculum. Copper sprays in late fall
or early winter to control fruit brown rot caused by Phytophthora citrophthora are also eective
against Septoria spot.
The traditional taxonomy of Septoria, accommodating more than 2000 species, is confused
as it has been based on few and conserved morphological characters. Moreover, it has been
largely dependent on host data, and most species are not restricted to a single host. However,
during the last years, the taxonomy of Septoria has been revisited using a polyphasic approach
including both multilocus DNA sequencing and morphological characters. A more robust
classication system is now available [35, 36]. In view of the worldwide distribution of S. citri
and its status as a quarantine pathogen in some countries, it would be interesting to examine
the genetic variability of Septoria populations associated to Citrus in dierent citrus-growing
areas of the world in the frame of this new classication system.
4.3. Greasy spot and other cercosporoid diseases
Several species of cercosporoid fungi have been associated with leaf and fruit spot dis-
eases of Citrus spp. Two of these diseases are particularly serious, Greasy spot, caused by
Zasmidium citri (Z. citri)-griseum (sexual morph Mycosphaerella citri), and Phaeoramularia fruit
and leaf spot, caused by P. angolensis, a fungus of quarantine concern for the European and
Mediterranean Region.
Figure 8. Typical symptoms of Septoria spot on a lemon fruit (courtesy S.O. Cacciola and A. Pane).
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
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Symptoms of greasy spot appear as yellow to dark brown to black lesions occurring rst on
the underside of mature citrus leaves. As the lesions develop on the underside of the leaves,
they become darker, and a corresponding chlorotic spot appears on the upper leaf surface.
Symptoms dier among citrus species. On highly susceptible species, such as lemon, spots
are diuse and tend to remain yellow, while on grapefruit, which is somewhat less suscep-
tible, lesions are less diuse, more raised and darker. On mandarins and ‘Valencia’ oranges,
which are much more tolerant, lesions are smaller, brown to black and much more raised.
Aected leaves fall prematurely from the tree during the fall and winter, resulting in reduced
tree vigour and yield. Beside defoliation, the disease causes a rind blemish on fruit which has
been referred to as greasy spot rind blotch. Greasy spot rind blotch signicantly reduces the
marketability of fruit for fresh consumption and is a serious problem especially on grapefruit
but can also occur on oranges and other citrus. Greasy spot was rst reported in Florida and
is now endemic in all citrus-growing areas of the Caribbean Basin [37]. It also occurs in Texas
but does not cause serious damage, probably because of a drier climate. Similar diseases
of citrus have been observed in Argentina, Australia, China, Brazil, Egypt, Japan, Korea,
Morocco, Spain and Italy. However the causal agent is not always Z. citri-griseum or has not
identied. In a recent study, four Zasmidium species have been recognized on Citrus, namely,
Z. citri-griseum, which has a worldwide distribution and a wide host range, and the three
Asian species Zasmidium fructicola, Zasmidium fructigenum and Zasmidium indonesianum [38].
During the last years, an epidemic outbreak of a foliar disease closely resembling greasy spot
has been observed in some citrus-growing areas of western Sicily (Italy). Symptoms appear
on mature leaves and range from those typical of greasy spot (Figures 9 and 10) to black dots.
Premature leaf drop occurs and causes heavy defoliation of the tree.
Figure 9. Symptoms of foliar greasy spot on the upper leaf surface of sweet orange (courtesy S.O. Cacciola and A. Pane).
Citrus Pathology16
The analysis of the fungal community using an amplicon metagenomic approach has revealed
that Mycosphaerellaceae were the dominant group of fungi, in both symptomatic and asymp-
tomatic leaves, and were represented by the genera Ramularia, Mycosphaerella and Septoria,
with about 44, 2.5 and 1.7% of the total detected sequences [39]. The most abundant sequence
type was associated to Ramularia brunnea, a species originally described to cause leaf spot in
a plant of the family Asteraceae. Surprisingly, none of the detected sequences clustered with
reference species currently reported as possible causal agents of greasy spot. Results are not
conclusive and the aetiology of this emerging disease is still unresolved.
5. Bleeding cankers caused by Botryosphaeriaceae and
Phomopsis/Diaporthe
Botryosphaeriaceae and Phomopsis/Diaporthe spp. are known to cause cankers on a variety of
woody hosts including citrus. Formerly this disease of citrus was known as Dothiorella can-
ker or Dothiorella gummosis because the pathogens most often isolated were Dothiorella spp.
However, recent studies have shown that the disease is caused by a complex of fungal species,
of which the most common belong to the family Botryosphaeriaceae and to a lesser extent the
genera Phomopsis/Diaporthe.
On citrus trees, cankers are found prevalently on trunk and main branches. The canker exudes
a reddish gum, giving it a bleeding, water-soaked appearance. Symptoms may also include
wilt of shoots and branches, sometimes with dead leaves still aached. Two types of fruiting
bodies (perithecia and pycnidia) can be found on cankers, which are the sexual and asexual
Figure 10. Symptoms of foliar greasy spot on the lower leaf surface of sweet orange (courtesy S.O. Cacciola and A. Pane).
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
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stage of these fungi, respectively. They produce the infective spores and appear as tiny black
bumps protruding from the bark. Pycnidial spores that are far more frequently observed in
nature than perithecial spores ooze out in a ribbon-like gelatinous matrix and are usually dis-
seminated by rain splash. Botryosphaeriaceae and Phomopsis/Diaporthe gain entrance into the
host through both wounds and natural gaps in bark continuity.
Species of Botryosphaeriaceae (Botryosphaeriales, Dothideomycetes) are cosmopolitan. Most of
them have a wide host range. On citrus, like other fruit trees, Botryosphaeriaceae are found
especially on stem and woody branches. In recent studies, the taxonomy of this group of
fungi has been radically revised using multigene phylogenetic analysis, and presently the
family comprises 22 recognized genera [40], including Diplodia, Botryosphaeria, Neofusicoccum,
Dothiorella, Neoscytalidium, Macrophomina, Lasiodiplodia and Sphaeropsis, just to cite a few of
them. Also the taxonomy of Diaporthe and its asexual morph Phomopsis has been revised on
the basis of phylogenetic and molecular data, and new species associated as endophytes with
citrus have been described [41, 42]. The report as a new disease of shoot blight, associated with
sooty cankers and gummosis, caused by Neoscytalidium dimidiatum in top-worked ‘Tarocco
Scirè’ trees on sour orange rootstock is an example of the radical change in classication sys-
tem and nomenclature of these groups of fungi. The same disease, in fact, was already known
as Hendersonula branch wilt as the causal agent was originally identied as Hendersonula
toruloidea [43]. Botryosphaeriaceae and Phomopsis/Diaporthe spp. are associated with citrus not
only as endophytes but also as saprobes as well as latent pathogens.
Biochemical and genetic stimuli, resulting from environmental changes inside the hosts (changes
in host physiological conditions or microbial equilibrium) or outside the host (climatic changes
or extreme environmental events), trigger these fungi to change their lifestyle from endophytic
to pathogenic. Therefore these fungi can be regarded as opportunistic fungal pathogens, and the
management of diseases they cause is based essentially on preventing environmental stresses.
In particular, an agronomical means to prevent the disease is to avoid water stress by reducing
the time intervals between irrigations. Surgical removal of infected bark does not restrict the
expansion of cankers, and copper treatments are only partially eective to reduce the inoculum
on the tree. They can be recommended to protect top-worked stumps and prevent shoot blight.
6. Wood rots
Wood rots are caused by a wide variety of wood-degrading microorganisms and are charac-
terized by decay and discoloration of wood of the trunk, large branches and main roots. Most
wood-degrading fungi are Basidiomycetes that on living trees can cause two major kinds of
decay: brown and white rots. Although wood-decay fungi play an ecologically important role
as primary biotic decomposers of wood in forest ecosystems, they can cause economic losses
in cultivated orchards by contributing to the premature ageing and the structural failure of the
trees. It can also aect young trees as a result of abiotic stress, such as severe frosts and sun
burning of branches exposed by heavy pruning. Most wood-decay fungi penetrate through
wounds, although a few of the root-infecting species can enter the unwounded surface directly.
Citrus wood rot is a chronic disease occurring endemically on old trees in most citrus-growing
areas of the world. Although this disease is not a major constraint for the citrus industry, it can
Citrus Pathology18
contribute to the deterioration of orchards because aected trees show a premature ageing, a
progressive decline in vigour and reduced productivity. A direct eect of wood decay is the
breakage of scaold branches due to loss of wood strength. Moreover trees show symptoms of
leaf chlorosis and twig dieback. The incidence of the disease is high in more than 40-year-old
orchards, and its severity depends on environmental conditions and susceptibility of the citrus
species and cultivars. In particular, lemon trees are signicantly more susceptible to wood
decay than other types of citrus, including orange, grapefruit and tangelo. In addition, the dis-
ease incidence seems to be correlated with the intensity and paern of precipitations. As far as
the Mediterranean region is concerned, Fomitiporia mediterranea (F. mediterranea) was found to
be the most common white wood-roing fungus of citrus [44–46], whereas Fomitopsis sp. was
associated with brown rot [47]. Other nonidentied basidiomycetous fungi showing genetic
anity with Phellinus and Coniophora were occasionally recovered from decayed wood.
F. mediterranea is a ubiquitous and polyphagus species, commonly found also on other fruit,
ornamental and forest tree species, such as hazelnut, olive, kiwi, locust tree and privet. On
grape, it is associated to ‘Esca’ disease. It is assumed that most infections in citrus orchards
originate from airborne basidiospores germinating on large pruning wounds of trunk and
main branches. Basidiospores produced by basidiomata germinate with relative humidity
over 90% and are dispersed by wind. Usually, basidiomata (Figure 11) emerge from the bark
after the wood of trunk or branches has been extensively colonized by the fungus (Figure 12).
In Southern Italy, the analysis of F. mediterranea internal transcribed spacer (ITS) sequences
revealed a high level of genetic variability, with both homozygous and heterozygous
Figure 11. Symptoms of wood rot caused by Fomitiporia mediterranea on Citrus sp. (courtesy S.O. Cacciola and A. Pane).
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
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genotypes. This and the high frequency of basidiomata in old commercial orchards conrm
the outcrossing nature of reproduction in F. mediterranea and the primary role of basidio-
spores in the dissemination of inoculum. Prevention is the only way to manage wood rots.
Trees should be kept healthy and vigorous. Large pruning cuts, or other wood-exposing
injuries, should be avoided, especially during wet periods. Proper management guidelines
include sanitation of pruning cuts with mastics to prevent the penetration of the pathogen.
7. Phytophthora diseases
The all-inclusive term “Phytophthora diseases” indicates a complex pathology which is caused
by soilborne species of Phytophthora and is recognized as a major fungal disease of citrus
almost universally. Phytophthora species aack citrus plants at all stages and may infect all
Figure 12. Basidiocarp of Fomitiporia mediterranea (courtesy S.O. Cacciola and A. Pane).
Citrus Pathology20
parts of the tree, including roots, stem, branches, twigs, leaves and fruits. There are several
forms (facies) of Phytophthora diseases including root rot, foot rot (Figure 13) (also known as
Phytophthora gummosis, trunk gummosis or collar rot), fruit brown rot (Figure 14), twig and
leaf dieback (often indicated collectively as canopy blight) and rot of seedlings (beer known
as damping o of seedlings). Trunk gummosis and root rot are the most serious facies of this
group of diseases, and after the pandemic outbreak of the nineteenth century and the con-
sequent widespread use of resistant rootstocks, they are regarded as endemic diseases in all
citrus-growing areas of the world.
At least ten species of Phytophthora have been reported to aack citrus in the world, but the
commonest species in commercial citrus orchards of the Mediterranean region are P. citroph-
thora and P. nicotianae [48]. The laer is the dominant species and is usually associated to root
rot, while P. citrophthora is frequent in old plantings and is commonly associated to trunk
Figure 13. Trunk gummosis caused by Phytophthora citrophthora on a citrus tree (courtesy S.O. Cacciola and A. Pane).
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
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gummosis. Other species found occasionally include Phytophthora citricola, Phytophthora cacto-
rum, Phytophthora hibernalis and Phytophthora syringae. The last two species are found during
winter months solely because of their low-temperature requirements.
Recently, Phytophthora meadii, which is new for the Mediterranean region, was recorded in
rhizosphere soil of poed ornamental citrus plants in southern Italy, using a very sensitive
amplicon metagenomic approach with genus-specic primers [49, 50]. Both P. citrophthora
and P. nicotianae are polyphagous.
Interestingly, however, genetic analyses of a worldwide collection of P. nicotianae isolates from
dierent hosts, including agricultural crops and ornamentals, revealed that isolates from cit-
rus clustered together and constituted a separate group regardless of their geographic origin
[51–53]. This result is indicative of host specialization and suggests that P. nicotianae popula-
tion associated to citrus has been very likely spread worldwide with infected nursery plants.
Temperature is a major ecological factor aecting seasonal uctuations of P. citrophthora and
P. nicotianae and their distribution. P. nicotianae prefers warmer temperatures, and in the
Mediterranean region, it is not active in winter, producing chlamydospores which allow the
pathogen to survive in unfavourable conditions. By contrast, P. citrophthora is not inhibited
by low temperatures, and during fall and winter, it can cause brown rot outbreaks. Another
epidemiological dierence between the two species is the ability of P. citrophthora to produce
sporangia on fruits, which are the sources of the secondary inoculum. This ecological feature
explains sudden epidemic explosions of brown rot, following persistent rainfall. Neither of
the two species forms sporangia on the gummy cankers at the base of the trunk. As far as it is
known, P. citrophthora does not reproduce sexually, and P. nicotianae reproduces sexually only
occasionally, since only A1 mating type is found in most citrus orchards.
Figure 14. Sweet orange fruit with symptoms of brown rot caused by Phytophthora citrophthora (courtesy S.O. Cacciola
and A. Pane).
Citrus Pathology22
Sporangia produced in the most supercial soil layer (0 to about 30 cm depth), on contact with
air, are the main source of inoculum. Natural infections are most frequently caused by zoo-
spores and occasionally by direct or indirect germination of sporangia through a germ tube or
by releasing zoospores, respectively. Production and germination of sporangia are inuenced
by temperature and soil water potential. Their dissemination is mostly by water splash and
occasionally by wind, within water droplets. The zoospores are motile and can swim short
distances by agellar movement or can be carried over longer distances by soil water. They
swim towards roots, as they are aracted by root exudates, and encyst upon contact, germi-
nate and penetrate fruits, leaves, shoots and green twigs directly.
Grafting on resistant rootstocks, such as sour orange, is the most practical and widely used
means to control Phytophthora gummosis. The rootstocks that are substituting sour orange
in the Mediterranean region, following the epidemic spread of Citrus tristeza virus (CTV),
to which sour orange is very susceptible, are mainly citranges, hybrids of trifoliate orange
(P. trifoliata) and sweet oranges, such as ‘Troyer’, ‘Carrizo’ and ‘C-35’ citranges. Generally
speaking, rootstocks resistant to P. citrophthora and Phytophthora gummosis are also resis-
tant to P. nicotianae and root rot, with few exceptions (e.g. ‘Carrizo’ citrange is resistant to
Phytophthora gummosis but susceptible to root rot, while trifoliate orange is resistant to both
facies of the disease).
Brown rot is both a preharvest and postharvest decay of citrus fruit. Infected fruit shows a
typical leathery brown rot with indistinct edges and has a characteristic rancid smell. With
high moisture in the environment, white furry mould forms on the fruit surface. Infections
cause the fruit to drop prematurely and occur especially on fruits hanging in the lower part
of the tree canopy, with rain splash. Epidemic explosions are more frequent in citrus orchards
where trunk gummosis is endemic. The incubation period of the disease is 7–10 days at 10°C
but may be longer with lower temperatures. Asymptomatic infected fruits can infect healthy
fruits even after harvesting, during transportation and storage.
Two major breakthroughs in the implementation of integrated management strategies
of Phytophthora diseases were the launch of systemic fungicides with specic activity
against Oomycetes [54–58] and the development of selective media for direct isolation of
Phytophthora spp. from soil and infected tissues. Serial dilutions of soil suspensions on a
selective medium became a very popular method for monitoring the amount of Phytophthora
inoculum in the soil. Monitoring was used to study seasonal uctuations of Phytophthora
populations to manage the irrigation as well as to schedule chemical treatments and evalu-
ate their eects [59–62]. The rationale of these strategies is the assumption that a direct cor-
relation exists between the amount of inoculum and the incidence and severity of root rot.
Although a very sensitive molecular method based on real-time PCR with specifc primers
was developed for the detection of P. citrophthora and P. nicotianae in soil [63], soil dilution on
a selective medium in Petri dishes still remains the preferred and most widely used standard
method for quantitative determination of Phytophthora inoculum. The quantity of inoculum
determined with this microbiological method is expressed in terms of colony-forming units
(CFU)/g or cm3 of soil. The threshold intervention level in bearing citrus orchards is between
10 and 30 CFU/cm3 of soil, but ideally a zero-tolerance threshold would be requested for
nursery stocks to be sold. More details on Phytophthora diseases may be found in many com-
prehensive reviews [48, 64–66].
Major and Emerging Fungal Diseases of Citrus in the Mediterranean Region
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23
8. Concluding remarks
Over the last decade, there have been a number of publications dealing with molecular studies
on fungal pathogens of citrus, focusing particularly on their identication and genetic diver-
sity. In particular, new species of Colletotrichum, Botryosphaeriaceae and Mycosphaerellaceae
have been described, and signicant progress towards a new, unied phylogenetic classi-
cation system of these groups of fungi has been achieved, for which substantial advances
were obtained by multigene sequencing and phylogenetic analysis. However, there have been
fewer publications related to other molecular aspects such as the mechanisms underlying
the host-pathogen interactions. With the development of next-generation sequencing tech-
niques and the availability of whole-genome sequences, molecular studies are expected to get
insight into the biology, pathogenicity and ecology of well-identied pathogens as well as the
aetiology of diseases whose causal agent is still undetermined. Moreover, diversity studies
will provide epidemiological information such as distribution, virulence and genetic struc-
ture of pathogen populations. The results of preliminary applications of these new molecular
techniques to the study of citrus diseases, such as Phytophthora diseases and greasy spot-like
diseases, are very promising.
Author details
Khaled Khanchouch1, Antonella Pane2, Ali Chriki1 and Santa Olga Cacciola2*
*Address all correspondence to: olgacacciola@unict.it
1 Laboratory of Genetic, Department of Life Sciences, Faculty of Sciences of Bizerte, University
of Carthage, Tunisia
2 Department of Agriculture, Food and Environment (Di3A), University of Catania, Catania,
Italy
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