MAL SECCO DISEASE OF CITRUS: A JOURNEY THROUGH A CENTURY OF RESEARCH - sipav
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001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 523
Journal of Plant Pathology (2011), 93 (3), 523-560 Edizioni ETS Pisa, 2011 523
INVITED REVIEW
MAL SECCO DISEASE OF CITRUS: A JOURNEY THROUGH
A CENTURY OF RESEARCH
F. Nigro, A. Ippolito and M.G. Salerno
Dipartimento di Biologia e Chimica Agro-Forestale ed Ambientale, Università degli Studi “Aldo Moro”,
Via Amendola 165/A, 70126 Bari, Italy
SUMMARY first name (“Poros’s disease”), soon spreading to Pelo-
ponnesus, Crete, Eubea and Thessaly (Sarejanni, 1935,
“Mal secco”, an Italian name meaning “dry disease”, 1939). In Italy, MSD was first reported in 1918 in the
is a severe tracheomycotic disease of citrus caused by district of Messina (eastern Sicily), apparently following
the mitosporic fungus Phoma tracheiphila (Petri) the introduction of infected plants from Greece (Rug-
Kantsch. et Gik. It appeared in 1894 in two Aegean gieri, 1949). The distinct symptomatology of the dis-
Greek islands, from which it spread almost to the whole ease, characterized by desiccation of twigs, branches, or
Mediterranean basin and the Black Sea. Due to its high the whole plant, suggested its extant name, “mal secco”
susceptibility, lemon is the most damaged citrus species. (“dry disease”) (Savastano, 1925), a denomination ever
Disease damage consists of substantial reduction of the since adopted internationally.
quality and quantity of the crop, mainly due to the diffi- In 1925, after the bewilderment for the tremendous
culties of controlling the disease and the replacement of damages suffered by the eastern Sicilian citrus industry
susceptible valuable cultivars by others which are less (primarily lemon), the Ministry of Italian National
vulnerable, but have low productivity and scarce fruit Economy entrusted Lionello Petri, head of Royal Plant
quality. Control of mal secco disease has relied on a Pathology Station of Rome, with the task of investigat-
number of diverse chemical and nonchemical strategies, ing the causes of the widespread decline of lemon plants
but is still faced with efficacy problems. Host resistance in the Messina district. The pathogen was identified as
remains a most desirable goal, but it will not be ulti- Deuterophoma tracheiphila (Petri, 1929a), following
mately achieved until the genetic basis of resistance to P. which a research station (“Osservatorio”) was estab-
tracheiphila are not fully elucidated. The present paper lished at S. Teresa Riva (province of Messina) with the
reviews the different aspects of citrus mal secco as stud- financial contribution of the “Messina’s Camera Agru-
ied worldwide over almost a century of research, from maria”, with the aim of studying in loco the biology, epi-
the first appeareance of the disease in Italy (1918) to demiology and control of the fungus. Notwithstanding
date. Milestones and pitfalls about the symptomatology, the efforts to keep MSD under control, it soon spread
aetiology, host-parasite relationship, diagnosis, epidemi- to the other main lemon-growing areas of Sicily, reached
ology, and control are discussed in a historical perspec- continental Italy, affecting the groves of Calabria, Cam-
tive, emphasizing the advancements in knowledge. Fi- pania, Apulia and Lucania (southern Italy), Latium
nally, some issues and challenges are highlighted that (central Italy), Liguria (northern Italy), and crossed
need to be more comprehensively addressed prior to again the sea to land in Sardinia.
deployment of effective disease control measures. The current geographical distribution of MSD com-
prises the east coast of the Black Sea (Georgia) and all
citrus-growing countries of the Mediterranean Basin,
HISTORY, GEOGRAPHICAL DISTRIBUTION AND except for Morocco, Portugal and Spain. Its occurrence
ECONOMIC IMPACT in Yemen has not been confirmed (EPPO/CABI, 1997;
EPPO/OEPP, 2007).
“Mal secco” (MSD) is a severe vascular disease of cit- Besides lemon [C. limon (L.) Burm. f.], the MSD
rus caused by the mitosporic fungus Phoma tracheiphila pathogen infects, with a relevant economic impact, oth-
(Petri) Kantschaveli et Gikachvili. It appeared in the er citrus species, such as cedar (C. medica L.), lime (C.
second half of 19th century (1894) in the Greek Aegean aurantifolia Christ.), bergamot (C. bergamia Risso),
islands of Chios and Poros, from which it derived its chinotto (C. myrtifolia Raf.), sour orange (C. aurantium
L.), rough lemon (C. jambiri Lush) and Volkamerian
Corresponding author: F. Nigro
lemon (C. volkameriana Ten. et Pasq.). The erratic field
Fax: +39.080.5442911 behaviour of the disease, makes its damages difficult to
E-mail: nigrof@agr.uniba.it estimate. In fact, one of the typical characteristics of001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 524
524 Mal secco disease of citrus Journal of Plant Pathology (2011), 93 (3), 523-560
MSD is the alternation of years with high or very high stripes on the affected organs. Ruptured epidermis per-
incidence with years in which infections are less severe mits the observation of pycnidia by the naked eye or
or nearly negligible. Furthermore, besides the direct with a low magnification lens. As reported by Grasso
damage to the plants, which can be crippled, if not and Perrotta (1978), pycnidia were produced on a num-
killed, and suffer heavy yield losses, there are indirect ber of species and hybrids belonging to the family Ru-
damages originating from: (i) the presence in the or- taceae, with the exception of C. myrtifolia, C. paradisi,
chards of plants of different age and susceptibility be- Fortunella sp. and Severinia buxifolia.
cause of the replacement of dead trees, which makes Double infections can be observed in malsecco-dis-
lemon production heterogeneous and lowers its qualita- eased trees. For instance, acervula of Colletotrichum
tive standard; (ii) the higher costs due to disease con- gloeosporioides (Penz.) Sacc., which are readily distin-
trol. According to a realistic evaluation, in the absence guished because of their arrangment in concentric rings
of MSD it would be possible to double the average Ital- (Fig. 3B) often occur on desiccated shoots in Sicily,
ian lemon production (Salerno and Cutuli, 1977). whereas the presence of Epicoccum granulatum Penzig
According to Ruggieri (1953), in the years 1918-1953 on different citrus species was recorded in Georgia
MSD has destroyed in Sicily no less than 12,000 ha of (Shumakova and Grube, 1957).
lemon groves, whereas in the Turkish district of Mersin According to Ruggieri (1956), MSD symptoms show
(Icel), the disease has killed about 20,000 lemon plants a seasonal fluctuation. The first signs of infection gener-
in 15 years (Karel, 1956). In the same Turkish area, Ak- ally appear and become more intense in spring-early
teke and Karaka (1977) recorded an average annual summer, to recede in the hight of summer and winter.
yield loss of 12.3%, which is much less than the Greek Although the appearance of symptoms and their more
estimates, that registered a 50-60% drop in the yield, or less rapid course depend from various factors related
with an average loss of 70, 45, 54, and 53% in the dis- to the age, vegetative stage and susceptibility of the
tricts of Patras, Temeni, Alissos and Chania, respectively host, the environmental conditions and the virulence of
(Thanassoulopoulos and Manos,1992). the pathogen’s strains, there are no doubts that the lo-
calization of primary infections play an important role
on their evolution. Infections starting from the canopy
THE DISEASE of adult plants generally progress slowly towards the
base, so that many years elapse before the plant dies. In
MSD induces a range of specific symptoms (Fig.1-3), the meantime, the plant reacts by producing sprouts
not all of which occur consistently in the different forms from the still uninfected branches or suckers from the
that characterize the disease. The first symptoms usually crown, which will be also infected. By contrast, when
appear on the leaves of the uppermost shoots, which the infection proceeds upwards from the base of a shoot
display a slight discoloration of the primary and the sec- or branch, the time-course of the disease is quite rapid
ondary veins. The leaves then turn yellow and fall, most- as a quick wilting of whole shoots or branches can en-
ly without the petioles that persit on the shoots (Fig. sue, accompanied by falling of the fruits and defoliation.
1C). These often show a chlorotic condition of the api- Exceptionally, wilted fruits and leaves remain attached
cal part, sometimes only on one side, while retaining a to the branch, as the rapid course of the disease pre-
normal green color in the basal part. Sometimes the vents the formation of the abscission layers.
shoots turn brown. Newly infected shoots show a yel- When the pathogen infects the outermost woody
low or pink-salmon to reddish discoloration of the rings of large roots or at the crown, symptoms appear-
wood, which occurs also in the wood of the main and ance involves just a sector of the host, less frequently the
secondary branches, as well as in the trunk, where the entire plants. However, within a short time, the plant
pathogen is advancing. A progressive basipetal desicca- dies, a condition called “mal fulminante” (sudden
tion of shoots, branches, and trunk follows and, finally, death). If the infection starts from rootlets, as it fre-
the whole plant may die (Fig. 1A). Generally, in the first quently happens in young nursery plants but also in
stages of infection, there is no clear-cut separation be- bearing trees in the grove, P. tracheiphila may remain
tween green and desiccated tissues. While the impact of segregated for many years in the inner wood layers. In
MSD increases witht the age of the plant, its severity is this instance the course of the disease is initially very
higher in young subjects. slow. However, as soon as the pathogen reaches the
A specific MSD trait is the occurrence of small, black most external woody rings the disease progresses very
and globose pycnidia of the pathogen (Fig. 3B) that can rapidly and the plant shows symptoms similar to those
readily be observed from the end of autumn on 1- to 2- produced by “mal fulminante” (Carrante, 1938; Cutuli,
year-old slowly desiccating shoots or suckers. Their 1972). In this case, instead of the characteristic salmon-
presence elicits the detachment of the epidermis from like color, the withering young shoots and main branch-
the underneath tissues, which is followed by penetration es display a browning of the innermost woody cylinder.
of air, resulting in the apperance of long silver-gray Wood discoloration becomes progressively more intense001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 525
Journal of Plant Pathology (2011), 93 (3), 523-560 Nigro et al. 525
Fig. 1. Symptoms caused by Phoma tracheiphila infections. A. A completely desiccated susceptible cv. Femminello lemon (fore-
ground) and a resistant cv. Monachello (background). B. Symptoms of “mal nero” caused by natural root infection of sour orange
rootstock. Note the discoloured triangular wood section expanding to a necrotic stripe on the cortex. C. Symptoms on the leaves
of a lemon shoot. The first symptoms usually appear on apical leaves as vein chlorosis. Symptomatic leaves often fall without the
petiole. D. Vascular vessels colonized by fungal hyphae. This mycelium produces phialo- and blastoconidia that move acropetally
with the transpiration stream.
in a downward direction until it acquires a blackish hue. onto susceptible rootstocks such as sour orange. Re-
Black-discoloured wood has a characteristic smell of cently, severe cases of “mal nero” on mandarin (cv. Cas-
overripe melon. Sometimes it happens that in corre- sar) and sweet orange (cv. New Hall), have been report-
spondence of a trunk sector, a lengthwise stripe of cor- ed from Tunisia (Hajlaoui et al., 2007).
tex appears necrotic and remains firmly attached to the According to Stepanov and Shaluishkina (1952)
underneath necrotic woody tissue (Fig. 1B). This partic- fruits and seeds of diseased lemon trees may be invaded
ular syndrome, described in detail by several authors by P. tracheiphila (Fig. 2). When unripe lemon fruits are
(Savastano and Fawcett, 1930; Carrante, 1938; Ruggieri, infected, they show partial or total yellowing of the peel,
1940), is called “mal nero” (black disease). The two syn- depending on the age of the infection, whereas ripe
dromes, i.e. “mal fulminante” and “mal nero”, can also fruits turn dark yellow to reddish. Diseased fruits nor-
be shown by resistant citrus species if they are grafted mally show signs of withering and fall to the ground.001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 526
526 Mal secco disease of citrus Journal of Plant Pathology (2011), 93 (3), 523-560
Fig. 2. Mal secco symptoms on lemon fruits. A. Extensive necrosis of the pericarp in the peduncular area. Infected fruits are gene-
rally smaller and tougher than the healthy ones. B. Discoloured seeds from infected fruits (upper row) compared with seeds form
healthy fruits (lower row). C. Vessels of infected fruits, showing various degrees of browning, those of the central axis in particu-
lar, depending on the age of the infection.
However, when infected branches desiccate rapidly the genus (Deuterophoma Petri). Althouh this finding was
fruits remain attached, showing necrosis of the pericarp repeatedly confirmed by L. Petri and endorsed by other
around the calyx (Fig. 2A), which may extend to the researchers (Savastano and Fawcett, 1930; Carrante and
equatorial zone and even further. Such fruits eventually Ruggieri, 1947), it remained for some time a controver-
mummify on the tree. Infected fruits show a red-brown- sial issue. For instance, Gassner (1940) attributed the ae-
ish discoloration of the vascular bundles which is more tiology of MSD to Phoma limoni Thum., which he con-
intense at the basal end but may also be seen in the up- sidered as a synonym of D. tracheiphila; an opinion
per end (Fig. 2C). This symptom is not specific as it may harshly opposed by Petri (1940). On the other hand,
occur also in fruits not affecd by MSD, as in the case of Pasinetti (1942) excluded the pathogenic role of D. tra-
endoxerosis (Cutuli and Salerno, 1998). Seeds of infect- cheiphila attributing the disease to unfavourable environ-
ed fruits are darker than those from healthy fruits, mental conditions. This hypothesis did not go very far.
mainly in the chalaza zone (Fig. 2B). Besides the lemon, The genus Deutherophoma was established to set a
fruits and seeds of other susceptible citrus species can difference from Sclerophoma von Höhn, which is char-
also be infected (Ippolito et al., 1987a, 1992). acterized by endogenous spores, contrary to the sup-
posed exogenous origin of D. tracheiphila pycnoconidia,
which are produced by budding. However, also Petri’s
THE PATHOGEN new genus was at the center of a controversy, as that be-
tween Petri himself (Petri, 1934) and Klebahn (1933),
Initially, the cause of MSD was erroneously attributed who erected the sub genus Blastophoma as a synonym of
to bacteria, which are common on citrus and many other Deuterophoma, maintaining that the pycnoconidia of
fruit trees (Savastano, 1923), then to C. gloeosporioides Sclerophoma were of both endogenous and exogenous
(Petri 1926, 1926a, 1927, 1927a, 1929), until the agent origin. A decade later Ciferri (1946) found that the mal
was ultimately identified by Petri (1929a) as secco pathogen had the characters of the genus
Deuterophoma tracheiphila, the type species of a new Bakerophoma Diedicke and proposed the new combina-001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 527
Journal of Plant Pathology (2011), 93 (3), 523-560 Nigro et al. 527
tion Bakerophoma tracheiphila (Petri) Ciferri. Shortly af- cheiphila in artificial media observing that the maximum
terwards, Goidanich and Ruggieri (1947), then Graniti fungal growth occurs between 20 and 25°C and that, in
(1955) amended the description of Deuterophoma sensu the same temperature range, pycnidia are formed more
Petri (1929a). Graniti, in particular, studied the mor- rapidly. At lower temperatures (10°C) pycnidia reach a
phology of the pycnidial and hyphal spores of D. tra- larger size and are profusely produced at 15°C. Pycno-
cheiphila, concluding that the fungus had characters dif- conidia germinate between 5 to 30°C, with an optimum
fering from those described by Petri as, for instance, the at 25°C. At 30°C, the growth of the germ tubes ceases.
mesondogenous origin of pycnoconidia. Following a Growth inhibition was observed at 0°C and 30°C, but
further re-examination of the fungal morphology with after 10 days at 30°C the fungus resumed its growth,
the electron microscope, Ciccarone and Russo (1969) when grown at 20°C. The color of the mycelial mat
confirmed that D. tracheiphila is a phyalidic species with changes in function of the incubation temperature, i.e.
meristogenous pycnidia provided with an ostiole and a white at 5°C but dark-grey at 25°C. On the whole, these
neck, and proposed its transferring to the genus Phoma results were in agreement with those by Stepanov
Saccardo. Later on, Ciccarone (1971) illustrated the ra- (1950), but not always with those by Petri (1939).
tionale whereby the fungus, according to the decision of As to the provenance of the pathogen, Ruggieri
the VIII Congress of Botany, had to be named Phoma (1948) agreed with Petri (1930) in hypothesizing its ori-
tracheiphila (Petri) Kantschaveli et Gikashvili, a binomi- gin from Asia Minor, as suggested by the disease pro-
al already used in the past (Kantschaveli and Gikashvili, gression from east to the west (Chios, Poros, Crete, and
1948) and provided the amended description of the Sicily), and to the south (Palestine) (Petri, 1930). More-
fungus. An English description of the same fungus, still over, it was also hypothesized that during this spreading
under the obsolete name of D. tracheiphila Petri, was the pathogen increased its virulence (Petri 1930).
shortly afterwards produced by Punithalingam and Hol- The natural occurrence of two different races of P.
liday (1973). tracheiphila was first reported by Petri (1930a, 1939).
Finally, it is worth mentioning that electron micro- Later, Baldacci (1950) labelled the two races as “DPR”
scope observations by Lo Giudice et al. (1982) revealed (colonies with dematiaceous mycelium, producing pyc-
that the septa of the fungal hyphae are monoporic with nidia and red pigment) and “PD” (colonies with dema-
a simple structure, a characteristic feature of the Phy- tiaceous mycelium, producing pycnidia but no red pig-
lum Ascomycota. Magnano di San Lio and Graniti ment). A third avirulent race (Petri 1939; Scrivani,
(1987) investigated the nuclear condition of P. tra- 1954), that usually appears after 2 or 3 subcultures on
cheiphila reporting that: (i) the mycelium is consitued by agarized media (Salerno and Perrotta, 1966; Messina,
mono- and plurinucleate cells; (ii) young hyphae and 1988) was called “R” (colonies without dematiaceous
apical cells are generally plurinucleate, whereas pycnidia mycelium, producing abundant red pigment but no py-
and conidiogenous phialide cells are uninucleate; (iii) cnidia). Race “DPR”, unlike “DP”, seems to occur
pycnoconidia and free phialoconidia are mostly wherever MSD is present. Contrary to the above views,
mononucleate; (iv) anastomoses occur among both hy- Goidanich and Ruggieri (1948) sustained that P. tra-
phae and conidial germ tubes. cheiphila is monotypic, and that the color and other
Although there are no doubts that P. tracheiphila is morphological traits vary in relation to different factors.
the causal agent of MSD, it should be kept in mind that Only the production of the red pigment, though in vari-
other pathogens can produce similar symptoms (Saler- able amounts, is a constant character of the fungus.
no, 1959). For example, Ruggieri (1946), reported the Salerno and Perrotta (1966) investigated the cultural
occurrence of vascular wilts of citrus due to Verticillium characteristics of some P. tracheiphila populations from
albo-atrum Rein. et Bert., and several other authors Sicily. All fungal isolates produced the red pigment and
pointed out the constant association between MSD and fluidized peptone gelatin, indicating that they belonged
infections by other fungal and bacterial pathogens, such to the “PDR” or “chromogenic” race (Petri, 1930b).
as C. gloeosporioides in Italy and Israel [see among the However, measurements of pycnidia and phialoconidia
others, Baldacci and Garofalo (1950); Reichert and conformed more with those characterizing the “PD” or
Chorin (1956)], Pseudomonas syringae Van Hall in “non chromogenic” race (Petri, 1930a; Baldacci, 1950).
Turkey (Chapot, 1963) and Epicoccum granulatum in These findings were confirmed by De Cicco and Luisi
Georgia (Shumakova and Grube, 1957). (1977), who studied 67 fungal isolates from different ar-
After repeated sub-culturing on agarized medium, P. eas and hosts from the Mediterranean basin. After-
tracheiphila loses some of the characters exhibited soon wards, Magnano di San Lio and Perrotta (1986) exam-
after isolation from infected tissues, in particular, the ined 600 P. tracheiphila isolates from Sicily, recovering
ability to produce pycnidia soon after the first or second non-chromogenic strains from a single plant near Paler-
subculturing. By contrast, repeated transferrings do not mo. These strains were similar to those of Baldacci’s
inhibit the production of phialides and phialoconidia. (1950) race “DP”, which had been found in the same
Salerno (1964) investigated the behaviour of P. tra- area. Studying isolates from lemon, orange, tangerine001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 528
528 Mal secco disease of citrus Journal of Plant Pathology (2011), 93 (3), 523-560
and grapefruit, Kantschaveli et al. (1976) distinguished anamorphic and teleomorphic taxa retrieved in BLAST
four different forms of the fungus, based on morpho- searches, revealed a close relationship between P. tra-
logical characters, as well as on carbon and nitrogen as- cheiphila and Leptosphaeria congesta (Balmas et al.,
similation and pH requirements. 2005a). However, the low number of fungal isolates
Colonies of P. tracheiphila growing on agarized media analysed (36) and the weak confidence value of the
show variant sectors, characterized by change in color, branch node did not lead the authors to deduce
different growth rate, and mycelial pattern. This vari- anamorphic/teleomorphic connection between the two
ability was partly attributed to the eterocariotic condi- species (Balmas et al., 2005a).
tion of the fungus (Graniti, 1969), which could deter- Comparable results were obtained by Ezra et al.
mine the separation of genetically different nuclei. (2007), who examined a small population of the fungus
However, conidiogenous cells of pycnidia and free (22 isolates) collected in Israel from various citrus
phialides are homocariotic. Then the nuclei of conidia species and growing areas. The isolates exhibited similar
produced by a single phialide would come from the morphological characteristics when grown in vitro. The
same ancestor, thus excluding that the variability occur- only difference was the inability of some isolates from
ring in the mitosporic and monocytogenetic lineage is lime and sour orange to express the red-orange pig-
due to heterocariotic dissociation. Moreover, consider- ment. The arbitrary primed polymerase chain reaction
ing the high frequency of the variant sectors in P. tra- (apPCR) profiling showed very similar patterns, regard-
cheiphila, it seems hazardous to hypothesize that this less of the fungal isolates examined, the different citrus
phenomenon is due to mutations or mitosis aberrations. species, the different locations and the different tissues.
Rather, it is reasonable to speculate that the variability Comparison of ITS1-5.8S-ITS2 sequences confirmed
of cultural characteristics in the monoconidial lineage the results of apPCR, and no significant differences
has an extranuclear origin (Jinks, 1966; Burnett, 1968). were found among the different isolates. This morpho-
Chromogenic and non-chromogenic variants show logical and genetic homogeneity would suggest that the
indistinguishable electrophoretic banding patterns of Israeli fungal population probably descends from a
total mycelial proteins and isoenzymes (catalase, es- common ancestor.
terase, glucose-phosphate isomerase), as determined by The analysis of a larger number of isolates from Italy
polyacrylamide gel electrophoresis (PAGE) of proteins and Greece, further confirmed the high homogeneity in
extracted from mycelium of pure cultures grown in liq- P. tracheiphila population. In fact, several isolates were
uid medium (Cacciola et al., 1986). Electrophoresis of identical over a ITS sequence 536 bp long, whereas only
mycelial extracts could however help in the identifica- two showed differences consisting of 2-4 nucleotide
tion of strains that differ in their capacity to produce substitution. A comparison of these sequences with
pigments or do not produce pycnidia (EPPO/OEPP, those deposited in GenBank revealed that 10 of 44 iso-
2007). lates tested showed only 2% sequences variation. More-
Despite the wealth of data available on the high vari- over, analysis of selected isolates, with or without se-
ability of phenotypic characters, such as colony mor- quence variation, confirmed a relationship with fungi
phology, pigmentation, and virulence, some recent pa- belonging to the Leptosphaeria sensu stricto group
pers suggest that such differences are not correlated (Grasso, 2008). The use of the amplified fragment
with the genetic variability of the fungus, as determined length polymorphism (AFLP) technique allowed a cer-
by molecular methods. Based on the analysis of ran- tain differentiation among P. tracheiphila isolates, al-
domly amplified polymorphic DNA (RAPD), mi- though no relationships were found with the geographic
crosatellite markers and sequencing of the internal tran- origin, cultural characteristics, and virulence (Grasso
scribed spacer (ITS) region of the nuclear rRNA genes, and Catara, 2006). By using the fAFLP variant of this
Balmas et al. (2005a) inferred phylogenetic relationships technique, in which primer labelled with a fluorophore
among isolates of P. tracheiphila, suggesting that the at 5’ terminus are used in the selective amplification
Italian population of the fungus is represented by a step, better results were recently achieved, although no
clonal lineage. In fact, the results obtained with RAPD clear-cut relationship with the geographic origin or oth-
and microsatellite markers showed that the Italian iso- er characters of the pathogen were found (Russo et al.,
lates of the fungus are genetically homogeneous, pro- 2011).
ducing identical patterns upon amplification with all The pathogenicity of P. tracheiphila has been investi-
primers tested. Accordingly, ITSI-5.8S-ITS2 sequences gated on a large number of isolates (142), collected
of all P. tracheiphila isolates were highly conserved (98- from different areas in the Mediterranean basin and var-
100% identity along a 544 character alignment). A ious natural hosts, by means of artificial inoculation of
neighborjoining analysis of P. tracheiphila ITS sequences sour orange, sweet orange and lemons of cvs Femminel-
in comparison with those of other Phoma species (P. lo and Monachello. Results of these trials led to conclu-
glomerata, P. esigua, P. betae, P. cava, P. fimeti, P. lingam, sion that there is no significant variation in the patho-
P. medicaginis) and with alignable sequences from genicity of P. tracheiphila, nor any apparent specializa-001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 529
Journal of Plant Pathology (2011), 93 (3), 523-560 Nigro et al. 529
tion (De Cicco and Luisi, 1977; Luisi et al., 1979, um and excised tomato plants, Graniti (1957) reported
1979a). that many of the symptoms reported by Scrivani (1954)
As mentioned, in artificial culture P. tracheiphila pro- were not caused by the phytotoxic activity of the
duces abundant red pigments of various hues, that form pathogen but by the cultural liquid. The same author,
crystalline aggregates on the surface of the hyphae and however, pointed out that the cultural liquid in which
are scarcely diffusible in the medium. Among them, the the fungus was grown, induced stronger and sometimes
anthrachinone derivatives helmintosporin and cinodon- different symptoms as compared with those described
tin have been identified (Quilico et al., 1952). More re- by Scrivani (1954). Some time later, Surico and Jacobel-
cently, a third pigment was found, chrysophanol, that lis (1980) studied the effect of cultural conditions on the
has a yellow colour and is probably responsible for the production of phytotoxins by P. tracheiphila obtaining
red-carrot or orange tonality that fungal colonies show the best results with static cultures exposed to light. The
on various artificial media (Ballio et al., 1979). P. tra- toxic activity of cultural liquids was maximum after 11-
cheiphila produces also humus-like substances, both in 12 days at 27°C, few days before the colonies had
artificial culture and during the colonization of citrus reached the maximum growth, to decrease afterwards, as
plants (Dzneladze, 1975), and the presence of flavins the mycelium underwent lysis. To evaluate the toxic ac-
and carotenoids has also been ascertained (Dzneladze, tivity of the cultural filtrates of the pathogen, testing ex-
1974). Culture filtrates of the fungus have an inhibitory cised or whole young tomato plants seemed to be more
effect on Citrus infectious variegation virus (CVV) and appropriate than citrus seedlings. The role of phytotoxic
on Tobacco mosaic virus (TMV), due to a polysaccharide compounds in the virulence of P. tracheiphila will be dis-
moiety and, to a lesser extent, a protein (Grasso et al., cussed in the heading “Toxins and Pathogenesis”.
1970; Grasso and Davino, 1974).
In artificial media P. tracheiphila produces enzymes,
e.g pectinolytic and inducible cellulosolytic enzymes HOST-PARASITE RELATIONSHIP
(Cx) as shown by Graniti (1969) and Cacciola et al.,
(1990). Evola et al. (1973) found the same, and showed Xylem colonization, wood discoloration, and gum
that the fungus is able to produce constitutively pectin- production. Once vascular tissues are infected, P. tra-
methyl-esterase (PME), and b-glucosidase. The same cheiphila spreads within the xylem vessels and move
authors, evaluated the effect of three different growth acropetally with the transpiration stream. The fungus
temperatures (12, 19 and 26°C) on enzyme production, then emerges from xylem vessels and colonize the
reporting that a higher activity of polygalacturonase neighbouring vascular tissues (Fig. 1D) thus inducing
(PG), trans-eliminase of polygalacturonate (PGTE), the sectorial symptoms seen in the wood. As a conse-
PME and macerating enzymes (MA) was evident at quence of xylem clogging, due to the presence of the
12°C, while b-glucosidase and CX were more active at fungal hyphae and the reaction of the host (gum pro-
19°C and 26°C, respectively. duction), the water and solute transport is compromised
The presence of phytotoxins in the cultural filtrates and water-stress symptoms appear. However, the wilting
of P. tracheiphila was first detected by Russian re- associated with MSD can only be in part justified by
searchers (Polyakov and Shumakova, 1951; Orshan- vessel occlusion. Electron microscopy observations
skaya, 1952), who investigated also the most favourable showed that only a thin layer of gum-like substances is
conditions for toxin production (Polyakov and Shu- visible inside the xylem vessels of infected plants (Mag-
makova, 1954). It was later shown that these toxic com- nano di San Lio and Perrotta, 1979; Bassi et al., 1980).
pounds derive from the lysis of the fungal mycelium and Perrotta et al. (1979a, 1981) investigated and dis-
that both lysis and toxin accumulation in the medium cussed the pathogenesis of P. tracheiphila in relation to
are higher at 23°C and above (Shumakova, 1964). xylem colonization. According to their findings, the
Studies on the production of toxins by P. tracheiphila fungus moves inside the vessels as passively transported
were also pursued by Italian scientists since the early spores, reaching the leaves before they show disease
1950s. Scrivani (1954) carried out a series of investiga- symptoms; moreover, it emerges from the vessels
tions on the occurrence of toxic metabolites in liquid through their punctuations, colonizing the neighbour-
culture of P. tracheiphila in Czapek medium amended ing xylem tissues. The rate and extent of xylem colo-
with corn-meal extract. The diluted cultural filtrates, ob- nization proved to be directly related to symptoms
tained by growing the fungus on this medium, were tox- severity and to the virulence of the different fungal
ic to lemon and tomato cuttings. The same author strains, and was a pre-requisite for symptoms appear-
proved also that the two pigments produced by race “R” ance. Moreover, the relative water content of the leaves
sensu Baldacci (1950) and identified by Quilico et al. did not show significant reduction until the wilt ap-
(1952) as anthraquinone derivatives were insoluble in peared, whereas an increase in the electrolyte leakage
water and were not phytotoxic. In a series of investiga- was observed in the early stages of disease development
tions carried out a few years later, using Scrivani’s medi- (Magnano di San Lio et al., 1992).001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 530
530 Mal secco disease of citrus Journal of Plant Pathology (2011), 93 (3), 523-560
Direct evidence of xylem impairment and water of these enzymes occurred in advanced disease stages,
transport alteration by P. tracheiphila was provided by these authors excluded a role in the stages correspon-
Raimondo et al. (2007), who ascertained an increase of ding to infection and vessel colonization. This conclu-
hydraulic resistance in stems and leaves of infected sour sion was supported by the lack of correlation between
orange seedlings. In particular, in infected leaves the the in vitro pectinolytic and cellulosolytic activity and
pathogen determined extensive clogging of the veins, the virulence of different isolates of the pathogen on
due to the progressive digestion of the interconduit pit sour orange seedlings (Pacetto and Grasso, 1974). More
membranes, which decreased the water potential recently, Cacciola et al. (1990) and Natoli et al. (1990)
threshold for the penetration of air into functioning showed that pectinolytic enzymes produced by P. tra-
conduits, thus facilitating the spread of the pathogen in cheiphila induce electrolyte leakage in the tissue of sour
the tissue. The time course of depression of leaf water orange leaves.
status and gas exchange was much more rapid when the
stem was infected, as compared with the values meas- Toxins and pathogenesis. Since the early studies on
ured when a leaf was inoculated (Raimondo et al., MSD, it was hypothesized that toxic substances pro-
2007), although the relationship between hydraulic con- duced by P. tracheiphila could play a role in pathogene-
ductance of the two organs and the leaf gas exchange sis (Petri, 1930). Several attempts to demonstrate the
were qualitatively similar (Raimondo et al., 2010). presence of toxins in the lymph or extracts from infect-
As mentioned, young infections confer a pink-salmon ed wood were successful (Kiyashko, 1951; Akhvlediani,
discoloration to the wood which with the time becomes 1958; Graniti, 1969), but the chemical nature of these
darker up to black, as in the facies of the disease known substances was not determined. Surico et al. (1981)
as “mal nero” (Figs. 1B and 4). The origin of these dis- studied the relationships between the degree of P. tra-
colorations was extensively discussed in the past. Petri cheiphila virulence and the in vitro production of toxins.
(1930, 1930a) attributed them to pathogen-produced When the phytotoxicity of culture filtrates was assessed
pigments absorbed by the walls of the vessels and of the on sour orange leaves, the two parameters (phytoxicity-
woody parenchyma cells, then diffusing into the gum virulence) were positively correlated, not so when ex-
masses of the xylem. However, histological observations cised tomato plants were used as indicators. In this case,
and the results of artificial inoculations with P. tra- the toxicity of filtrates was directly correlated with the
cheiphila isolates with different ability to form pigments in vitro growth of the fungus, rather than with the viru-
in artificial culture, cast doubts on the fact that wood lence of the tested isolates. Thus it appears that P. tra-
discolorations were due to diffusing fungal pigments. It cheiphila can produce more than one phytotoxic sub-
was thought, instead, that the discoloration was the ex- stance, with different selectivity. Subsequently, Pennisi
pression of the gummosis reaction of infected vascular et al. (1988) addressed the same topic, and found a sig-
elements (Goidanich and Ruggieri, 1948, 1953; Bugiani nificant correlation between virulence and electrolyte
et al., 1959). However, gums that, like those observed in leakage in sour orange leaf discs treated with toxins pro-
mal secco-infected tissues, may show a shade of pink, duced in liquid medium by hyper- and hypo-virulent
are produced in the wood of citrus plants infected by isolates of P. tracheiphila. The elution of toxins from a
other pathogens (Salerno, 1959) or damaged by cold, or sepharose column produced two protein peaks, but on-
wounded. Therefore, gum production is an aspecific re- ly the one with lower molecular weight showed biologi-
sponse of citrus to injuries of various origin. Later inves- cal activity. Pennisi and Graniti (1987) measured the
tigations (Ballio et al., 1979; Matarrese Palmieri et al., electrolyte leakage from leaf tissues, in the attempt to
1979; Perrotta et al., 1981) confirmed that the discol- correlate symptoms with changes in the permeability of
oration of infected wood is mainly associated with the cell membranes. Yellow leaves close to abscission
gums accumulating in xylem tissues, and ultra-structural showed more important changes than those with milder
examination of these tissue showed that the gums origi- symptoms, whereas in symptomless leaves from infected
nate from the alteration of the primary cell wall and twigs the permeability changes registered were small.
middle lamella complex (Magnano di San Lio and Lo The converging observations of Magnano di San Lio et
Giudice, 1982; Cacciola, 1989). al. (1992), led to the hypothesis that alterations of cell
membrane permeability contribute to the water stress
Hydrolitic enzyme activity. Production of gums was syndrome associated with MSD.
also observed after artificial introduction of pectinolytic The phytotoxic activity of P. tracheiphila was also ex-
enzymes in the wood of sour orange shoots (Bugiani et tensively investigated in Israel. In the culture extracts of
al., 1959). Pacetto and Davino (1976) studying PME the pathogen Nachmias et al. (1977) found the presence
and Cx in citrus plants artificially and naturally infected of an extracellular glycopeptidic substance, that was
by P. tracheiphila, found that the activity of both en- phytotoxic and able to induce disease symptoms in
zymes was higher in extracts from infected xylem than lemon shoots. The toxin had an estimated molecular
in those from healthy tissues. Since the highest activity weight of 93 kDa and an isoelectric point of 4.3. The001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 531
Journal of Plant Pathology (2011), 93 (3), 523-560 Nigro et al. 531
carbohydrate moiety (29.5%) consisted of mannose, treated plants, possibily because of the toxin’s interfer-
galactose and glucose whereas the peptide moiety ence with the genetic control of chlorophyll biosynthe-
(36%) contained most of the common amino acids, as- sis. Finally, a compound identified as mellein (Parisi et
partate, glutamate, threonine and serine being the most al., 1993), and occurring at a very low concentration in
represented. 14C-labelled toxin was obtained by grow- P. tracheiphila culture filtrates, was not phytotoxic when
ing the fungus in the presence of radioactive amino tested on lemon leaves but, according to the authors, it
acids, and radioactivity was readily translocated to could have synergistic action with other phytotoxic
lemon cuttings. The detection of radioactivity in the metabolites produced by the pathogen.
leaves was correlated with the appearance of symptoms.
The toxin induced electrolyte leakage from plant tis- Mechanisms of resistance. This issue has been the
sues, reduced the transpiration rate, and inhibited the object of attention since the aetiology of MSD was es-
growth of callus of lemon cv. Eureka, at concentration tablished. Although in the early 1930s it was thought
which were ineffective on Shamouti sweet orange that resistance could be related with the number of
(Nachmias et al., 1977a). The toxin was then searched stomata occurring on the leaves, Rabinovitz-Sereni
for in infected plants, as evidence for its role in disease (1931) dismissed this notion as he was unable to find
development (Nachmias et al., 1979). From artificially such a correlation. The subsequent studies of Petri
infected leaves of rough lemon, a glycopeptide, very (1939, 1940), led him to conclude that the chemical
similar to the phytotoxic compound present in cultural properties of the xylem sap contribute to the resistance
filtrates, was isolated. Since this glycopeptide was de- of sweet orange to MSD.
tected in infected plants in a quantity that could justify Insofar as a structural type of resistance is concerned,
the production of symptoms, the authors concluded it is worth reporting both the Goidanich and Ruggieri
that this compounds had a role in pathogenesis and was (1947, 1947a) hypothesis about the histological and
classified as a “vivotoxin” (Dimond and Waggoner, functional modifications that take place following deep
1953), for which the name of “malseccin” was proposed infections by wounding, and the observations by Som-
(Nachmias et al., 1979). Subequently it was found that ma et al. (1979), according to which the resistance of
malseccin is a complex of glycoproteins with different lemon (cv. Monachello) observed in the first year post
molecular weight, whose major phytotoxic fraction, de- infection, is due to the formation of new wood in ad-
noted Pt60, has a molecular weight of 60 kDa. When vance of the spreading of the fungus. More recently,
the properties of mycotoxin Pt60 were examined, no se- Lanza et al. (1980) examined the wood anatomy and
quence homology was found with any known protein water conductivity of old and nucellar clones of some
(Fogliano et al., 1994, 1998). lemon cultivars in relation to their resistance to MSD,
Other Israeli studies showed that partially purified concluding that the different behaviour of the analyzed
preparations of the malseccin complex damaged the cultivars was not associated with the length and diame-
chloroplasts and inhibited the photosynthetic fixation ter of the vessels, nor with water conductivity. In previ-
of carbon in the leaves of rough lemon (Nachmias et al., ous studies, however, Paculija (1959) and Scaramuzzi et
1980). When the effect of partially purified Pt60 was al. (1964) associated the susceptibility to mal secco in-
evaluated on the viability of protoplasts of several Ru- fections with luxuriant vegetation. Finally, according to
taceae species and cultivars a differential sensitivity was Tokhadze (1971) the resistances to MSD seems directly
observed. Non-citrus species were more tolerant to the correlated with linked water and inversely with free wa-
toxin. Electron microscopy observations revealed severe ter content.
ultrastructural alterations of the cellular membrane, and Following the results of Luisi et al. (1979), who re-
the chloroplast bounding membrane and thylakoids ported that artificially inoculated mature leaves of dif-
similar to those observed in in vivo infected tissues (Ses- ferent resistant and susceptible cultivars exhibit the
to et al., 1990). Damage to chloroplasts and/or reduc- same level of susceptibility/resistance of mature plants
tion of photosynthesis since the first stages of MSD, has in the field, Bassi et al. (1980) and Perrotta et al. (1979,
been reported by several authors (Demetradze and 1979a, 1981) investigated the cytological and histologi-
Dzhanelidze, 1970; Demetradze et al., 1972; Kantschaveli cal changes of infected leaves of differently susceptible
et al., 1972; Uturguari et al., 1973). citrus species. The alterations more frequently encoun-
Other phytotoxic substances have also been identi- tered were: hyperplasia and hypertrophy of xylem
fied and characterized. A termostable, hydrophilic, low parenchyma cells, plasmolysis, gumming of the vessels,
molecular weight (350-700 Da) phytotoxin, acting as cell wall modifications, crushing of the vessels, derange-
de-coupling of the electron transport in chloroplasts ment of plastids, increased number of mitochondria. In
and inducing chlorosis in lemon leaves was identified the susceptible Femminello lemon, P. tracheiphila prolif-
and partially purified by Barash et al. (1981). Goliadze erated actively invading the vascular bundles, the xylem
et al. (1972), by treating lemon seeds and seedlings with parenchyma reacted hyperplastically and hypertrophy-
P. tracheiphila toxins obtained albinism in 57% of the cally, xylem tissues were disrupted and the necrosis ex-001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 532
532 Mal secco disease of citrus Journal of Plant Pathology (2011), 93 (3), 523-560
tended to the cambium, but the fungus was not restrict- gree of susceptibility to MSD, and a variable phenolic
ed. Instead, in the resistant Monachello lemon the content in the roots and leaves. However, irrespective of
pathogen progressed for a short distance from the site the lower or higher susceptibility to the disease, several
of infection and only within the injured tissues. The phenols (m-hydroxybenzoic, o-cumaric, and pherulic
cambium was unaffected and remained active. Similarly, acids) increased in response to P. tracheiphila infection.
Goliadze and Kerkadze (1971) observed in an artificial- Subsequently, Evola et al. (1976) investigated the effect
ly inoculated susceptible Georgian cultivar a necrosis of of some phenolic compounds and hesperidin on the en-
the cambium and a phloem and medulla significantly zymatic activity of P. tracheiphila, showing that the de-
larger than in the resistant Meyer lemon. velopment of the fungus was significantly inhibited in
As to the supposed role of gum deposits in the resist- substrates containing either one of the following: hes-
ance to MSD, it can be hypothesized that this substance peredin, m-coumaric, gentisic, ferulic, 4-hydrox-
can only slow down the spread of P. tracheiphila, since yphenylpyruvic and o-coumaric acid. Polygalacturonate
the fungus survives and sporulates in vessels filled with transeliminase, pectinmethyl-esterase, ß-glucosidase and
gum (Bugiani et al., 1959; Graniti, 1969). Nevertheless, polyphenoloxidase activity was reduced most consis-
gums may have a bearing in the transmission of infec- tently in the presence of hesperedin, whereas variable
tion from the leaves to the branches, as suggested by increase or reduction in activity were observed with the
Traversa et al. (1991) who found more gum than myceli- different phenolic compounds. Therefore, the conclu-
um in the main veins and petioles of artificially inoculat- sion was that there is no correlation between the com-
ed resistant Monachello and sour orange leaves, where- pounds exerting a inhibitory activity on the pathogen’s
as the mycelium was more abundant in the leaf veins of enzyme production and the accumulation of the same
susceptible Femminello. compounds following mal secco infections of sour or-
The chemical mechanisms of resistance, both pre- ange seedling (Salerno et al., 1971a).
and post-infection have attracted much attention. Ac- Catara et al. (1971, 1972, 1973), after testing the
cording to Egorova (1958), resistant lemon cultivars dif- fungistatic activity of phenolic extracts from healthy
fer from the susceptible ones (e.g. Novogeorgian plants and plants affected by infectious variegation or
lemon), because of the higher activity of peroxidases, exocortis, studied the post-infectional phenolic metabo-
the higher and different acid and alkaloid content, and lism, and observed that in sour orange and Feminello
the quantity of nitrogen and soluble alkali. Reduced lemon P. tracheiphila induced an accumulation of free
catalase and peroxydase activity and a higher ascorbic phenols 6-8 days post inoculation. The accumulation
acid content were reported in susceptible cultivars was higher in plants with the resistant Vaniglia orange
(Tsiklauri, 1972). However, according to Pacetto and interstock (Davino et al., 1974). Later, the same authors
Grasso (1969) and Pacetto and Davino (1980), it does examined the variations of free acid phenols during the
not seem that the activity of oxidative enzymes can be time course of MSD but did not reach ultimate conclu-
related to the degree of susceptibility to MSD. Goliadze sions on the role of these substances in the mechanism
(1960) refers of substances generically indicated as phy- of resistance to the disease (Davino et al., 1979, 1979a).
toncides, which he found active only in resistant lemon More recently, Reverberi et al. (2008) conducted an
cultivars and in the roots of Poncirus trifoliata. in planta and in vitro study on the role of the oxidative
In resistant citrus plants Ben-Aziz et al. (1962) found stress in the lemon-P. tracheiphila interaction, using the
two substances, one of which strongly inhibited the cvs Monachello, Interdonato, and Femminello, which
growth of P. tracheiphila in culture. In subsequent inves- are considered as resistant, partially resistant, and sus-
tigations (Ben-Aziz, 1967) some compounds of the ceptible to mal secco infections, respectively. When in-
flavone group were isolated from tangerine plants, oculated with P. tracheiphila, cv. Interdonato leaves
among which, nobiletin and tangeritin afforded a high showed chlorosis and necrosis and an increase in
fungistatic activity. Pinkas et al. (1968) isolated five lipoxygenase and glutathione peroxidase. Furthermore,
flavones from tangerine, four of which were active in extracellular proteins of P. tracheiphila infiltrated into
vitro against P. tracheiphila. Subsequently, Piattelli and the leaves induced lipoperoxide formation tenfold high-
Impellizzeri (1971) found no correlation between the er in cvs Interdonato and Femminello, and threefold
concentration of nobiletin, tangeritine and 5,4’-dihy- higher in cv. Monachello compared with the control,
droxy-6,7-8,3’ tetrametoxiflavon and resistance of some with Monachello reacting earlier. Results from in vitro
citrus species. experiments indicated that the amendment of the fungal
The role of phenolic compounds in the defence growth medium with lyophilized twigs and leaves of cv.
mechanism of citrus plants against mal secco infection Monachello stimulated the concentration of superoxide
was first investigated by Salerno et al. (1970, 1971a). dismutases, glutathione peroxidase, and catalase in the
Sour orange seedlings inoculated with the agents of viral mycelium. When lyophilized twigs and leaves of cv.
diseases known as concave gum and infectious variega- Femminello were added, the pathogen produced a high-
tion, and with the exocortis viroid showed a variable de- er quantity of hydrolytic enzymes, such as polygalactur-001_JPPInvitedReview_523 15-11-2011 17:35 Pagina 533
Journal of Plant Pathology (2011), 93 (3), 523-560 Nigro et al. 533
onase and laccase. After growth in malseccin-conducive (PAGE) is also recommended (EPPO/OEPP, 2007).
media, a high amount of monoamine oxidase was found
in the extracellular proteins of P. tracheiphila. The en- Conventional methods. Conventional detection pro-
zyme catalyzes oxidative deamination of primary cedures rely on the observation of symptoms in the
amines, leading to a toxic accumulation of H2O2 and field, the isolation of the fungus from infected tissues
NH4 in the host cell. Thus, the extracellular enzyme and a comparative study to establish whether its mor-
monoamine oxidase could be among the different com- phological characteristics matches those of P. tracheiphi-
pounds involved in MSD. la. Mal secco symptoms have been largely described
above. However, since the occurrence of symptomless
infections has been reported (Di Silvestro et al., 1988;
DIAGNOSIS Balmas et al., 2005; Russo et al., 2008), this possibility
should be taken in due consideration when propagative
Based on the symptoms shown by infected plants, material is tested. As mentioned, the presence of pycni-
one would assume that MSD diagnosis is easy. This is dia (Fig. 3B) on infected plants is a specific symptom of
why the early diagnosis techniques developed during MSD, and the in vivo characteristics of these organs are
the 1940s through the 1960s, were simple and readily very important for species differentiation. As stated be-
applicable, as exemplified by the use of substances such fore, pycnidia can be easily observed by the end of au-
as potassium hydroxide, sodium hydroxide, alcohol, tumn-winter on 1- or 2-year-old shoots which desiccat-
and ammonia on the woody cylinder of infected plants ed slowly. These fruiting bodies are scleroplectenchyma-
for the early detection of wood discoloration, tous, black at maturity, globose or more often rather
(Kantschaveli and Gikachvili, 1948; Orshanskaya, 1952, flattened, lenticular ostiolate, and measure 60-165 × 45-
1953; Fedorinchick, 1953; Sinitsyna, 1953; Bazzi and 140 mm. They occur rarely on the leaf stipules (Graniti,
Scrivani, 1954). 1963) and are formed primarily around the leaf scars or
P. tracheiphila is a quarantine pathogen of great con- in the cracks of wilted cortical tissues. Pycnidia then
cern for regional and national Plant Protection Services emerge in large and undefined areas of the wilting
worldwide, i.e. European and Mediterranen Plant Pro- shoots, which acquire a silver-gray colour originated by
tection Organization (EPPO), Asia and Pacific Plant the lifting of the epidermis detached from the underly-
Protection Commission (APPPC), Caribbean Plant Pro- ing tissue and the black shade of the fruiting bodies. Py-
tection Commission (CPPC), Comité de Sanidad Vege- cnidia are scattered or densely aggregated in small
tal del Cono Sur (COSAVE), Inter-African Phytosani- groups which coalesce, freely settled in the disrupted
tary Council (IAPSC), North American Plant Protec- and frayed cortical tissue.
tion Organization (NAPPO), Pacific Plant Protection One of the most complete and detailed in vivo de-
Organization (PPPO) which provide detailed informa- scription of P. tracheiphila structures (pycnidia, pycno-
tion for avoiding or restraining the spread of the conidia, phialides, and phialoconidia) was provided by
pathogen and for its correct identification. For APPPC, Ciccarone (1971), and will be briefly recalled here. De-
COSAVE, CPPC, and PPPO the recommended regula- veloping pycnidia are astomatous, but at maturity devel-
tory status of P. tracheiphila is in the A1 list, i.e., “a op a long neck 45-70 µm in diameter and up to 250 µm
quarantine pest not present in that area”. Instead, the in length. The necks are cylindrical or tendentially ob-
IAPSC and EPPO include P. tracheiphila in the A2 list, conical, quite often flared at the top, surrounded by a
as a “quarantine pest present in that area but not widely dense and very dark hyphal mat that gathers under the
distributed there, and being officially controlled” (EP- epidermis, cementing the fruiting bodies to the epider-
PO/OEPP, 2009; 2010). As mentioned, P. tracheiphila mis and together in groups. The necks are easily re-
does not occur in several citrus-growing countries of the moved with the epidermis, leaving behind widely and
EPPO region, like the Iberian peninsula, Corsica (insu- irregularly opened pycnidial bodies. The wall of mature
lar France) and Morocco, although no obvious climatic pycnidia consists of randomly arranged polygonal scle-
or cultural factors prevent its potential establishment in roplectenchymatous cells, and is of about the same
these uninfected areas. As the introduction of P. tra- thickness throughout. The surface of the pycnidial cavi-
cheiphila is a serious threat to lemon-growing areas, re- ty is uniformly covered by very small phialides (3-4.5 x
strictions on the movement of citrus propagating mate- 3-5.5 µm), irregularly saccular, widely conical or pyri-
rial are mandatory. To be effective, these provisions form, tapering apically in a very short neck, no more
must be supported by the availability of procedures for than 1 µm tall. They produce minute unicellular,
a reliable, quick, and sensitive diagnosis. According to mononucleate and sometimes binucleate, hyaline pyc-
the standard diagnostic protocol of EPPO, P. tracheiphi- noconidia (0.5-1.5 × 2-4 mm), with rounded ends,
la can be identified by conventional and molecular shortly ellipsoid, irregularly pyriform, and sometimes
methods. In some particular cases, the analysis of slightly curved. Conidia are sometimes extruded
mycelial proteins by polyacrylamide gel electrophoresis through the ostioles in whitish cirri. Free hyphae grow-You can also read
