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Diseases involving phloem restricted prokaryotic agents
Witches' broom disease of lime
Witches' broom disease is a lethal disorder of small-fruited acid lime trees in Oman. The disease was first studied and shown to involve an MLO in 1986 (Bové, 1986b; Bové et al., 1988). It has now reached the United Arab Emirates (Taher, personal communication, 1990). Chapter 16, which deals with Oman, describes the disease.
Stubborn
In view of the progress made in the study of stubborn in the Mediterranean and the Near East, and because of its wide distribution in these regions, the disease will be covered in some detail. See also Chapter 7.
Name and synonyms. The term stubborn was applied as early as 1921 to non-productive navel orange trees in California, as indicated by Fawcett, Perry and Johnston when they described the disease in 1944. Less commonly used names in California include acorn disease of oranges (Haas, Klotz and Johnston, 1944), stylar end greening, and blue albedo of grapefruits. In the country then known as Palestine, Reichert and Perlberger described little leaf disease of citrus in 1931. Citrus stubborn disease as known in California and little leaf disease as described in Palestine are identical.
The causal agent, Spiroplasma citri, infects and produces diseases in non-rutaceous hosts as well as in citrus (Caravan, 1980; Gumpf and Calavan, 1981). The name "stubborn" should be restricted to the disease in citrus.
Causal agent. The causal agent of citrus stubborn disease is the helical mollicute (mycoplasma) S. citri (Saglio et al., 1971a; Fudl-Allah, Calavan and Igwegbe, 1972: Fudl-Allah and Calavan, 1974) (see Figs 22 to 24). S. citri was the first spiroplasma to be characterized (Saglio et al., 1973) and for which Koch's postulates were fulfilled (Markham and Towsend, 1974).
Like other mollicutes, spiroplasmas require sterol for growth but they are unique in that they are motile and have a helical morphology (see Figs 22 to 24). Since the discovery of S. citri more than 30 other spiroplasmas have been identified, and at the present time they fall into 23 defined serogroups. S. citri is part of serogroup I. This group is further subdivided into eight subgroups, S. citri representing subgroup I-1 (Bové, 1984). Like other members of group I, S. citri has DNA with 26 mole percent Guanine plus Cytosine, and a genome size of 109 daltons. S. citri is serologically related only to spiroplasmas of group 1, and especially to those of subgroup I-2 (honeybee spiroplasma or Spiroplasma melliferum) and I-3 (corn stunt spiroplasma or Spiroplasma kunkelii). By DNA-DNA hybridization it shows relatedness not only to these spiroplasmas but also to those of subgroup I-8 (spiroplasma P40 or Spiroplasma phoeniceum). S. citri, S. kunkelii (Whitcomb et al., 1986) and S. phoeniceum (Saillard et al., 1987) are the only three sieve tube-restricted, plant pathogenic spiroplasmas known today. S. citri can be identified unambiguously by a number of techniques, including serology and analysis of its proteins by one- and two-dimensional PAGE (Mouchès and Bové, 1984). Spiralin (26 x 105 daltons) is the major protein of the S. citri membrane (Wroblewski, Johansson and Hjerten, 1977); it is specific to S. citri in that anti-spiralin monospecific immunoglobulins (IgGs) do not recognize proteins of other spiroplasmas. However, spiralin-like proteins are probably present in all spiroplasmas. The spiralin gene has been cloned in Escherichia coli, in which it is expressed (Mouchès et al., 1984), and its nucleotide sequence has been established (Chevalier, Saillard and Bové, 1990).
S. citri can be infected by three different viruses: SpV1, SpV2 and SpV3 (Cole, 1979) but not by SpV4, a virus specific to S. melliferum. The nucleotide sequence of the single-stranded, circular DNA of the filamentous SpV1 virus has been determined (Renaudin et al., 1987 and 1990).
S. citri can be cultured in relatively simple mycoplasma media (Bové, Whitcomb and McCoy, 1983). The optimum temperature for growth is 32°C. The smallest viable S. citri cell is a two-turn "elementary" helix that grows essentially by one end into a longer parental helix. A four-turn parental helix divides by constriction into two elementary helices. Longer parental helices undergo multiple divisions. Genome replication is not coupled to cell division (Garnier, Clerc and Bové, 1984). S. citri, like eubacteria, contains three DNA polymerases but, unlike eubacteria, its RNA polymerase is insensitive to rifampicin (Gadeau, Mouchès and Bové, 1982). Like all other mollicutes, S citri is insensitive to penicillin and other antibiotics interfering with bacterial cell wall synthesis. Data on the cellular and molecular biology of spiroplasmas have recently become available (Bové et al., 1989).
Host range. Symptoms of stubborn disease in the field are seen essentially on sweet orange and grapefruit. Table 7 lists other susceptible species, while Table 8 gives symptomless species. Tolerant rootstocks do not make susceptible scions tolerant. Susceptible species are sometimes symptomless carriers under cool temperature conditions.
In the southwestern United States of America many cultivated and wild non-rutaceous plant species were found to be naturally infected with S. citri (Oldfield and Calavan, 1980). In the eastern United States, S. citri-infection of horse radish causes brittle-root disease. In Cyprus, Morocco, Oman, the Syrian Arab Republic, Turkey and the United Arab Emirates, periwinkle plants (C. roseus) exposed to natural contamination in the field became infected with S. citri (see Figs 175 and 176).
In addition to natural infection, experimental transmission of S. citri to many non-rutaceous plants by leafhoppers has increased the host range of the spiroplasma (Oldfield and Calavan, 1980). For insect hosts, see the section below on transmission.
Symptoms of citrus stubborn disease.
Aspect of affected trees. These are slightly to severely stunted (Figs 144, 147, 148, 152 and 157), frequently with abnormally densely bunched and upright foliation. Excessive dieback of shoots and branches is present in severe cases (Fig. 144). Multiple buds develop resulting in mild witches' brooms.
Fruit symptoms. Affected trees have low yields. The proportion of symptomatic fruit on a tree is variable. Fruits can be small and/or lopsided (curved columella) (Fig. 168). They sometimes show colour inversion, the peduncular end becoming coloured while the stylar end remains green (Fig. 167). They are acorn-shaped when, at the peduncular end, the albedo is thick and the flavedo coarse while, at the styler end, they are respectively thin and smooth (Fig. 165). The vascular network in the albedo is sometimes prominent and the vascular bundles brownish-red. Blue albedo is sometimes observed (Fig. 169). The type of fruit symptoms may depend on the environmental conditions in which the trees grow.
Foliar symptoms. The presence of small, cupped leaves (Figs 149 and 150) is typical of stubborn, hence "little leaf" for the disease in Israel. Leaves can also have abnormally upright positions. internodes are short (Figs 147 and 163). Various types of mottle can affect the leaves (Figs 153, 154 and 158). The distal portion of certain expanding leaves can be pinched-in and yellowish-green (Fig. 155). This is of diagnostic help, especially on indicator plants. Under hot dry conditions (Iran, Iraq), leaves of certain shoots are palmate or cordate with pinched-in, yellow tips (Fig. 160). Such shoots (Figs 161 to 163) are highly diagnostic (Bové et al., 1984).
Trunk symptoms. With severely affected, mature trees grafted on sour orange there is sometimes pinholing (honeycombing) on the cambial side of the bark immediately below the bud-union line (Fig. 145). This symptom is common to both stubborn and tristeza diseases, and therefore can be misleading.
In the field, fruit symptoms are more useful than leaf symptoms for positive diagnosis. Culture of S. citri and ELISA (see next section) are most helpful in confirming diagnosis based on symptoms.
Indexing.
Use of indicator plants. Vigorously growing Madame Vinous sweet orange seedlings kept at temperatures close to 32°C (optimum growth temperature of S. citri) in the day and near 27°C at night are used as the indicator plants. Inoculum usually consists of young leaf patches, including the midrib (Caravan, Olson and Christiansen, 1972), but side grafts have also been used. Inoculation grafts are made directly below the indicator bud to be forced. Seven or more indicator seedlings should be used per tree to be indexed, together with healthy controls for comparison.
Symptoms to be observed are: slow growth; small cupped leaves; short internodes between leaves; and mottled leaves, with the distal portion being pinched-in and yellowish-green.
Detection of S. citri by culture and ELISA. There is very good correlation between symptom expression of stubborn in Mediterranean countries and the detection of the causal agent (S. citri) by culture and ELISA (Bové et al., 1984).
The plant materials used as a source are: seeds with various degrees of abortion, the peduncular ends of fruit axes and mature, mottled leaves from the summer flush of growth, collected in October under Mediterranean conditions.
For culture assay for S. citri the M1A or SP4 media of Whitcomb (1983) are used. Isolation of the stubborn spiroplasma is done by the filter method of Bové, Whitcomb and McCoy (1983). For assay using ELISA, the assay procedure using anti-S. citri IgGs, as described by Saillard and Bové (1983), is used.
Detection of S. citri by DNA probes, PCR and IC-PCR. Techniques more sensitive than ELISA and the culture assay are currently being developed and should be available soon. They are based on DNA hybridization with DNA probes, gene amplification by PCR and immuno-capture (IC) of the spiroplasma prior to PCR (IC-PCR).
Transmission. Natural transmission is through budwood, in highly varying percentages, and through leafhopper vectors (Caravan and Bové, 1989). The following leafhoppers transmit S. citri in California: Neoaliturus (ex-Circulifer) tenellus (sugar beet leafhopper see Fig. 35), Scaphytopius nitridus and Scaphytopius delongi. In the Mediterranean area, N. tenellus is present but N. haematoceps (ex-N. opacipenis - see Fig. 34) - also a sugar beet leafhopper and from which S. citri could be consistently cultured -is the major vector (Fos et al., 1986).
A much preferred host plant of N. tenellus and N. haematoceps is Salsola kali (Chenopodiaceae) (see Fig. 36 and Figs 170 to 174). This plant is widely distributed throughout the Mediterranean and the Near East. Its presence near or within nurseries or young orchards (Fig. 174) can provide the focus for epidemics of stubborn disease (see Chapter 7).
Geographical distribution. Citrus stubborn disease is widespread throughout the Mediterranean and Near East areas, and the southwestern United States of America. S. citri is present in at least the following countries or regions: Algeria, Cyprus, Egypt, France (Corsica), Greece, the Islamic Republic of Iran, Iraq, Israel, Italy, Lebanon, the Libyan Arab Jamahiriya, Mexico, Morocco, Oman, Saudi Arabia, Spain, the Syrian Arab Republic, Tunisia, Turkey, the United Arab Emirates and the United States of America (Arizona and California). In Iran the presence of stubborn correlates well with the presence of N. haematoceps and N. tenellus (see Map 2). In turn, the presence of the two leafhoppers is related to the presence of S. kali. A similar situation is observed in Cyprus, Morocco and the Syrian Arab Republic (see Map 3).
Economic importance. Stubborn disease is destructive in most countries that grow citrus under hot, dry, desert or semi-desert conditions. Fruit quality is inferior and many fruits are malformed. Production from diseased trees can be reduced by 50 to 100 percent. In California, 5 to 10 percent of sweet orange and grapefruit trees are estimated to be affected. In certain Mediterranean areas the damage is even more severe.
Greening
In view of the importance of greening for Near East countries and the threat it represents for the Mediterranean region, still free of the disease and its vectors, greening is described more thoroughly than some of the other diseases. The following description is an updated version of a recent European Plant Protection Organization data sheet on greening (EPPO, 1988).
Names and synonyms. Greening has received different names according to the country affected, but in each case the same type of Gram-negative bacterium is involved, even though strain differences may exist, in particular, heat-tolerant (Asian) and heat-sensitive (African) forms of the disease, as discussed below. The disease is known under various names: greening in Africa; huang longbing, i.e. yellow shoot disease, in China; likubin. i.e. Ieaf mottle disease, in Taiwan Province; leaf mottling in the Philippines; citrus decline in India and vein phloem degeneration in Indonesia.
Principal hosts.
Rutaceous hosts. The bacterium may persist and multiply in most Citrus spp., but most severe symptoms are found on sweet orange (C. sinensis), mandarin (C. reticulata) and tangelo (see also Tables 9 to 11).
The vectors are confined to Rutaceae, including both wild hosts (Clausena anisata, Vepris undulata) and commercial citrus varieties. Lemon (C. limon), lime (C. aurantifolia) and Murraya paniculata, a rutaceous plant often used for hedges or ornamental purposes (Fig. 218), are preferred hosts for Diaphorina citri.
Non-rutaceous host. Experimental transmission of the citrus greening bacterium from citrus to periwinkle (Catharanthus roseus) by dodder (Cuscuta campestris) has been achieved (Garnier and Bové, 1983).
Geographical distribution. The African (heat-sensitive) form of the disease has been reported from the Comoros, Ethiopia, Kenya, Madagascar, the Republic of South Africa, Swaziland and Zimbabwe, and more recently from northern Yemen. The Asian (heat-tolerant) form is present in China, India, Indonesia, Malaysia, Nepal, Pakistan, the Philippines, Taiwan Province and Thailand, as well as Saudi Arabia. Both forms of the disease exist in Mauritius, Reunion and southwestern Saudi Arabia (Bové and Garnier, 1984). The Mediterranean area and most of the Near East (e.g. the Islamic Republic of Iran) are still free from the disease. The infected area closest to the Mediterranean zone extends south of Mecca, along the Red Sea.
D. citri occurs throughout the Asian zone of greening (and also in Brazil and Japan, but in the absence of greening), while Trioza erytreae occurs throughout the African zone, in northern Yemen, and also in Cameroon and Saint Helena. Both insects occur together in Mauritius, Reunion and southwestern Saudi Arabia.
The citrus greening bacterium. Transmission of the greening pathogen by graft inoculation was first reported in China (Lin, 1956). Laflèche and Bové (1970) found that the disease was characterized by the presence of an apparently mycoplasma-like micro-organism, not a virus, in the sieve tubes of affected plants. However, when soon thereafter the agent of citrus stubborn disease was discovered (Igwegbe and Calavan, 1970) and found to be a mycoplasma (Fudl-Allah and Calavan, 1974; Saglio et al., 1971a) and more precisely a spiroplasma (Saglio et al., 1973), comparison between the greening agent and S. citri showed the greening agent to be different from a mycoplasma (Saglio et al., 1971b). Since then, considerable work has confirmed this view and shown the greening agent to be a bacterium with a peptidoglycan-containing membranous cell wall of the Gram-negative type (Moll and Martin, 1974; Garnier, Latrille and Bové, 1976; Garnier and Bové, 1977; Garnier, Danel and Bové, 1984a, b). The bacterial nature of the greening organism explains why penicillin treatment of infected plants results in symptom remission (Bové et al., 1980; Aubert and Bové, 1980). A report claiming culture of the citrus greening bacterium has appeared (Garnett, 1985). There is, however, at the time of writing (March 1993), no experimental evidence to show that the cultured organism is the greening bacterium.
As stated above, two forms of greening disease are known (Bové et al., 1974). One, in the southern part of Africa, is heat-sensitive, as symptoms do not develop in hot climates where temperatures above 30°C are reached for several hours a day. The other form is heat-tolerant and withstands high temperatures. It is present in China, India, Nepal, Southeast Asia and probably Pakistan. The Asian heat-tolerant form of the disease has been discovered in Saudi Arabia, and the African heat-sensitive form in northern Yemen (Bové and Garnier, 1984). When the African and the Asian forms of the greening bacterium were transmitted from citrus to periwinkle (Fig. 220) by dodder (Fig. 219) (Garnier and Bové, 1983), the African form remained heat-sensitive and the Asian form remained heat-tolerant in periwinkle as well as in citrus.
Monoclonal antibodies (MAs) to the citrus greening bacterium, and more specifically to Indian, Chinese and South African strains, have recently been obtained for the first time (Garnier, Martin-Gros and Bové, 1987; Garnier et al., 1991). By immunofluorescence on sections (Fig. 221) or ELISA, the MAs react essentially with the homologous strains, indicating that there are strain differences. Purification of the greening bacterium by immunoaffinity has been achieved (Villechanoux, Garnier and Bové, 1990).
Insect vectors. Under natural conditions the greening pathogen is transmitted in Africa, Madagascar and northern Yemen by T. erytreae (McClean and Oberholzer, 1965) and in Asia (including Saudi Arabia) by D. citri (Capoor, Rao and Viswanath, 1967). However, it has been shown experimentally that T. erytreae can transmit the Indian form of the greening agent (Massoniée, Garnier and Bové, 1976) and that D. citri is able to transmit the African form (Lallemand, Fos and Bové, 1986).
The two psyllid vectors of the greening organism are restricted to rutaceous hosts and especially citrus. They are citrus pests in their own right (Figs 212 and 213), in addition to being vectors of greening.
T. erytreae (see Fig. 37). The vector of the African form of the greening bacterium, the psyllid T. erytreae, behaves like the African form of the bacterium with respect to temperature sensitivity (Calling, 1973a; Schwarz and Green, 1970). It is very sensitive to extremes of hot, dry weather (the eggs and first instar nymphs being particularly vulnerable), and is favoured in cool, moist areas over 500-600 m above sea level, where citrus growth flushes tend to be prolonged. Green and Catling (1971) have used maximum saturation deficit as an accurate predictor of the geographical distribution of T. erytreae. Sex ratios fluctuate in the field, but females always dominate. Eggs are often laid along the leaf midrib and margins (Fig. 188). Nymphal development (five instars) takes 17 to 43 days. The temperature threshold for nymphal development is, apparently, around 10-12°C. There is no diapause. Developing nymphs live in concave depressions on the lower surface of the leaf, resulting in the presence of convex, gall-like bumps on the upper surface (Figs 189 to 193, 207 and 209). No such bumps are associated with D. citri.
Diaphorina citri (see the drawing on p. 75 and Fig. 37; Figs 210 and 211). D. citri has a short life cycle and high fecundity (Calling, 1970; Schwarz and Knorr, 1973). It is more prevalent in hot coastal areas. Pairing starts soon after emergence, the insects being most active during March to April in India (Pande, 1971) and May to June in the Philippines (Calling, 1970). Eggs are laid singly from March to May inside half-folded leaves of the buds, in leaf axils or other suitable places on the young tender parts of the tree. Females have a preoviposition period of about 12 days, and are capable of laying up to 800 eggs within two months. Eggs hatch within a period varying from three days in summer to 23 days in winter, and nymphs pass through five instars in 11 to 30 days. In dry periods the adults are numerous, but nymphs are usually absent. The complete life cycle thus takes from 14 to 48 days, and there can be up to ten overlapping generations per year.
The adults overwinter and can live for up to six months. They are very active and jump at the slightest disturbance. Nymphs will move away when disturbed, but normally lead a sedentary existence clustered in groups. Population fluctuations are closely correlated with the flushing rhythm of citrus trees, since eggs are laid exclusively on young flush points.
Economic importance Citrus greening is an extremely severe disease. In the Republic of South Africa, in 1965, fruit losses from the disease were 30 to 100 percent in individual orchards, many of which had subsequently to be abandoned or removed. Earlier outbreaks occurred in 1932-36 and 1939-46. Current annual losses are estimated at 35 million South African rends (Van den Berg, Van Vuuren and Deacon, 1987). In Mauritius, Reunion and Thailand, large areas of citrus cultivation have had to be abandoned (Calling, 1973b; Schwarz and Knorr, 1973). In the Philippines, mandarin production fell from 11 700 tonnes in 1960 to 100 tonnes in 1968 (Commonwealth Department of Health, 1982). In southwestern Saudi Arabia, sweet orange and mandarin practically disappeared during the 1970s (Bové, 1986a). In Southeast Asia, an FAO-UNDP programme has recently been established to try to control the disease.
Heavy infestations with T. erytreae cause leaf distortion and gall-like bumps on the upper surface of the leaves, but not the defoliation or dieback typical of D. citri, which can also cause serious damage to growing points, leading to dwarfing as well as lack of juice and taste in fruit. Heavy D. citri populations can cause blossom and fruitlet drop.
In the Transvaal, citrus is treated against greening by injection of tetracycline (up to 20 g per adult tree) with high-capacity compressors working at 10 kg/cm². Such methods have been tried out, but not widely used, in Asia. Dimethoate can be used against the vectors on orchards with low infection rates, while the highly toxic aldicarb is used in South Africa as a soil-applied biocide (Bové, 1986a). In Reunion, the vectors have been successfully controlled by the introduction of parasites - Tetrastichus dryi Waterston, from South Africa, for T. erytreae and T. radiatus Waterston, from India, for D. citri (Aubert, Bové and Etienne, 1980). In Saudi Arabia, however, T. radiatus is present but does not keep D. citri populations down to a low level (Bové, 1986a). T. erytreae, and especially D. citri, could probably establish and spread in Mediterranean and Near East countries with out difficulty. It should be remembered, too, that both these, psyllids have significant damage potential in themselves. Though biological control may be possible, there is no guarantee that it could keep populations to a sufficiently low level to prevent the transmission of greening. In view of its severity, it is essential to keep the disease (and its vectors) out of the Mediterranean basin and to prevent its spread in the Near East.
The situation in Brazil, and possibly other South American countries, is different. D. citri is present in Brazil, but greening itself has never been reported from North, Central or South America. Hence introduction of infected plant material, even without the vectors, would be most dangerous.
The rutaceous plant, Murraya paniculata, frequently used as an ornamental bush or hedge, is one of the best hosts of D. citri (Fig. 218). This plant can carry eggs or nymphs of the vector, thus its introduction into disease-and vector-free regions could be dangerous. Quarantine services should be advised.
General aspect of affected trees (Figs 177 to 179, 196, 197, 200, 204 and 205). Affected trees show: open growth, stunting, twig die-back, sparse yellow foliage and severe fruit drop. On certain trees and in certain countries (China), symptoms are seen initially on one limb of an affected tree (this is known as yellow branch aspect) (Fig. 217). Severe decline is seen mainly with Asian greening.
Symptoms on fruits (Figs 194 and 195). Some fruits are underdeveloped, lopsided and poorly coloured. Seeds are often aborted (Figs 195 and 201). The so-called "greening" fruit symptom refers to fruit which matures only on the side exposed to the sun, the unexposed side remaining a dull olive green.
Symptoms on leaves (Figs 180, 184, 187, 202, 203 and 214). Mottling and zinc deficiency-like symptoms are the most common and characteristic leaf symptoms. Mature leaves often show irregular patches between the main veins and the veins are often prominent and yellow.
Symptoms on trunk, limbs and shoots. No symptoms are apparent.
Histological symptoms. Localized zones of necrotic phloem are scattered throughout the vascular system of the leaf. Massive accumulation of starch in the plastics is seen, together with aberrations in cambial activity and excessive phloem formation.
Symptoms due to vectors. T. erytreae severely distorts leaves, which become stunted and galled as the nymphs induce depressions on the lower sides of the leaves. These are viewed as "bumps" on the upper sides (Figs 187, 189 to 193, 207 and 209). Leaves appear to be dusted with faecal pellets. D. citri stunts and twists young shoots, so that the growing tips present a rosetted appearance.
Leaves are severely curled (Fig. 212), and may be covered with honeydew (Fig. 211) and sooty mould, dropping prematurely.
Indexing and detection.
Biological indexing. Suspect material may be grafted on sensitive indicator plants - sweet orange seedlings are preferred. Inoculation should preferably be with pieces of mottled leaves. Because of the variable results in graft transmission, at least ten seedlings should be used for each tree to be indexed. After inoculation, the indicator seedling should be kept at 24°C (South African form) or 32°C (Asian form). The symptoms usually show up after four to five months. The presence of a specific fluorescent marker, gentisoyl glucoside (Feldman and Hanks, 1969) in greening-infected tissue has been used for indexing (Schwarz, 1968a, b).
Electron microscopy (see Figs 3 to 21). As there are no symptoms specific to greening, suspect trees have to be examined using electron microscopy to confirm bacteria in the sieve tubes (elongated, sinuous, rod-like structures 0.150.25 µm in diameter and several microns long, as well as a round form, 0.3-1.0 m in diameter). Similar structures have been seen in both vectors (Moll and Martin, 1973; Chen, Miyakawa and Matsui, 1973).
Serological identification. The MAs against the Indian and the Chinese strains of the citrus greening bacterium have been successfully used to detect the homologous bacterium in greenhouse-grown citrus and periwinkle, using immunofluorescence (Fig. 211) and ELISA (Garnier et al., 1987, 1991). However, the MAs against the greening BLO are too strain-specific to be suitable for general diagnostic work.
DNA probes. DNA probes for the detection of the greening BLO have recently been obtained (Villechanoux et al., 1992 and 1993). One of these probes is able to detect all Asian BLO strains tested, even when it is used at high stringencies of hybridization. At lower stringencies it also detects South African strains. The probe has been extensively used to detect the greening BLO in orchard trees in India.
Even though this review concerns essentially virus and virus-like diseases, some widespread and often encountered fungal diseases have been included.
Phytophthora gummosis or footrot
This disease is caused by soil fungi (Phytophthora citrophthora and Phytophthora nicotiana var. parasitica) which kill the bark of affected trees. This very serious disease is controlled by using resistant rootstocks to prevent the fungus from attacking the susceptible mandarin, sweet orange or grapefruit scion varieties.
Sour orange is a phytophthora-resistant rootstock, and this is the major reason why this species has been used so widely as a rootstock for more than 100 years. The benefit of using a phytophthora-resistant rootstock is lost, however, when the tree is budded too low, bringing the susceptible scion too close to the soil or, even worse, when the tree is planted too deep, with the bud-union becoming buried in the soil (Fig. 228). Phytophthora gummosis is also favoured when farmers put soil around the trunks and cover up the bud-union lines (Figs 224 to 226). Severe forms of gummy bark are associated with bark cracking and scaling (Figs 68 and 74) and these outer bark lesions may become ports of entry for the fungus (Fig. 68), especially when, in addition, the bud-union line is close to the soil.
Phytophthora gummosis is essentially a disease of the bark; only a thin layer of wood under the affected bark may be stained brown. The entire bark, not only the outer layer as in Rio Grande gummosis (see below) or scaly bark psorosis, is affected by the fungus. Unlike Rio Grande gummosis, there are no pockets of gum in the wood. The disease often begins at the soil level when a susceptible citrus species (scion or rootstock) is directly in contact with the soil (Fig. 230). Trunk lesions rarely extend higher than 35-40 cm from the ground or the bud-union line.
Good horticultural practices to protect trees from Phytophthora
Symptoms of phytophthora gummosis above ground are: dead areas of bark that remain firm; exudation of small or large amounts of gum, depending on citrus varieties and weather (Fig. 230); infiltration with gum and brown staining of a thin layer of wood and presence of gum between bark and wood (Fig. 228); and a subsequent drying and vertical cracking of bark.
In early stages, the decayed bark is firm and intact, but with age it becomes shrunken and cracked, shredding in lengthwise strips as it dries. Much gumming may accompany advanced stages of footrot, but because of the solubility of gum in rainwater this symptom is not always conspicuous. The bark remaining alive above the lesions often develops callus rolls that check further spread, especially in an upward direction. In some cases the disease appears to be arrested, only to resume extension at a later date. Ultimately, the lesion may encircle the trunk, killing the tree. However, the effects of footrot in the tops vary according to the extent of the trunk lesion. A characteristic development of footrot is that the tree dies irregularly, one side failing while the other side remains sound. In declining parts of the top, the foliage at first is hard and dull, assuming a light yellow cast, and eventually becomes distinctly yellow, especially along the midribs. These are nothing but the characteristic symptoms of acute starvation brought on by girdling of all or part of the trunk. Starvation patterns are not only seen in the foliage but also include reduction in fruit size, leaf drop and dieback, and a reduction in vegetative growth.
In the following cases footrot can also occur below ground, and soil must be removed from around the tree to expose the lesions.
The rootstock is phytophthora-susceptible. The order of susceptibility among rootstocks commonly used is, starting with the most susceptible: sweet lime, lemon, acid lime, sweet orange, rough lemon, Cleopatra mandarin, citranges, sour orange and P. trifoliata.
The bud-union line is below soil level. The rootstock trunk and part of the scion trunk are in the soil. Footrot lesions can develop on the rootstock, and on the scion part, if susceptible. This is the case with grapefruit and sweet orange, for instance.
Soil is put around the trunk, burying the bud-union line. Removal of the soil exposes phytophthora lesions (Fig. 228).
In the above cases, development of the pathogen, Phytophthora spp., requires that the soil in contact with the phytophthora-susceptible trunk be moist or wet. Soil moisture at or near saturation is most favourable for fungal growth, spore production and movement of zoospores. The fungus is very sensitive to moisture fluctuations and its activity ceases when soil dries out.
P citrophthora grows fastest when the temperature is near 25°C. P. parasitica has a higher optimum, near 30°C. A pH of 6.0-7.5 favours the growth and multiplication of the fungi. Control of phytophthora gummosis is through good horticultural practices. Trees should be planted on a heap of soil with the crown above soil level, not below. The scion bud should be grafted at about 15-20 cm above the crown of the seedling rootstock. Flood irrigation water should be prevented from reaching the trunk by building soil levees around the trees (see the drawings on P. 79).
Mal secco
Mal secco (an Italian name meaning dry disease) is a highly destructive disease of lemon trees. The causal organism is the fungus Phoma tracheiphila (Petri) Kantsch & Gik. (syn. Deuterophoma tracheiphila Petri). It is readily cultured and on potato-dextrose agar the mycelium produces abundant red pigments. A similar red-orange coloration is seen in xylem recently invaded by the fungus. This diagnostic coloration is revealed by cutting through the wood (Fig. 234) or peeling off the bark (Fig. 235). Mal secco affects primarily lemon, but also sour orange and citron trees. Susceptible rootstocks are rough lemon, sour orange and Troyer and Carrizo citranges. Femminello lemon is particularly susceptible, while Monachello, Interdonato and Santa Teresa show some resistance. The disease attacks trees of any age but is more severe on young ones. Infection by the fungus is either in the canopy or in the roots. In the first case, the leaves wilt, dry up and are shed, and die-back occurs (Figs 232 and 233). The pathogen proceeds slowly downwards from young shoots to branches, main limbs, trunk and roots, and it may take several years for the infection to reach the trunk. In Italy and Cyprus, infections can also occur in the roots. The fungus proceeds rapidly upwards, producing symptoms on the whole tree or only on one limb. The disease may develop so suddenly that the leaves dry up on the tree. In another type of root infection, the pathogen invades the inner xylem vessels without at first inflicting any apparent damage to the tree. Eventually, however, the pathogen reaches the external rings of the wood and then causes a sudden collapse of the canopy (Solel and Salerno, 1989).
Mal secco is widespread throughout the citrus-growing countries of the Mediterranean and Black Sea regions: Cyprus; France, but not Corsica; Greece; Israel; Italy, including Sicily; Jordan; Lebanon; the Libyan Arab Jamahiriya; the former Union of Soviet Socialist Republics; the Syrian Arab Republic; Tunisia; and Turkey. The disease is apparently not yet present in Iraq, Iran or the Arabian Peninsula.
Rio Grande gummosis
The causal organism of Rio Grande gummosis (RGG) (Caravan, 1961; Childs, 1978) has been identified only recently (Davis, 1980). In Florida, Godfrey (1946) and Childs (1953) had attributed the disease to an actinomycetous fungus. In Texas, Olson (1952) and Olson and Waibel (1953) attributed it to a Diplodia sp. In California, Calavan and Christiansen (1958) and Calavan (1961) suggested an unidentified basidiomycetous fungus. Calavan et al. (1962-1963) found two fungi commonly associated with the disease. One, the unidentified fungus of Calavan and Christiansen (1958), enters through pruning wounds and other injuries and invades the pith and heart wood. The other, Hendersonula toruloidea Nattrass, invades wounds and heat- or frost-injured areas (Caravan and Wallace, 1954). There is also some circumstantial evidence that the disease is related to a high concentration of chlorides in the soil (Childs, personal communication).
Based on the following observations, Diplodia natalensis Evans (syn. Physalospora rhodina Cke.) appears to be the causal agent of RGG, at least in Texas (Davis, 1980): D. natalensis was consistently isolated from RGG-affected tissue; symptoms of RGG developed in healthy tissue inoculated with pure cultures of D. natalensis and the fungus was subsequently reisolated, fulfilling Koch's postulates; the disease was transmitted with affected tissue, establishing the infectious nature of the disease; and disease development was arrested with applications of the fungicide benomyl.
The most obvious symptom of RGG is profuse gum production (Figs 236 to 240). The gum oozes out of vertical cracks in the bark (Figs 244 and 245) and runs down along the trunk or hangs down from the branches, stalactite fashion (Figs 236 to 240). At the time of initial gumming there is no scaling of the bark at the sites where the bark is split (Fig. 244). However, the first stage in the healing-over process is the sloughing of thin scales of dead outer bark. Then follows the development of scar tissue generated by the bark. Repair is only temporary and healed-over lesions may again start gumming and enlarging (Figs 241 and 242). In this way, lesions may pass through repeated cycles of recovery and relapse, in the course of which they progressively enlarge and expose more and more wood. In old, inactive lesions (Figs 240 and 243) the wood is exposed. The gum pockets may be located deep in the wood and the gum travels a considerable distance in and along the wood, so that gum pockets may exist well removed from the nearest active, gum-producing lesion. The bark scaling associated with the healing-over process and the sloughing of scales of dead outer bark can be pronounced (Fig. 243) but should not be confused with that of scaly bark psorosis (Figs 98 to 100) or "popcorn" symptoms (Fig. 101). Nor should RGG be confused with phytophthora gummosis or footrot. Table 14 helps to distinguish between RGG, scaly bark psorosis and phytophthora gummosis.
Grapefruit and lemon are more frequently affected than other varieties. In decreasing order of susceptibility these varieties are sweet orange, tangerine and sour orange, the last being practically immune. In one Somalian orchard, grapefruit trees were grafted on sour orange with the bud-union line high above the soil. There was abundant gumming on the grapefruit part of the trunk but none on the sour orange part. In lemon trees, gumming breaks out just above the bud-union and involves progressively more and more of the trunk. In grapefruit and orange trees, symptoms appear higher up the trunk and out on the larger branches. Trees on rough lemon rootstocks appear to gum more profusely than those on sour orange.
There seems to be a correlation between the first symptoms of RGG and the year when the grapefruit trees are pruned for the first time. It has been pointed out that the symptoms show up soon after the trees are first pruned. In Somalia, for instance, trees are pruned at six to seven years, and RGG appears when the trees are seven to eight years old. These observations correspond with results obtained in California, where it was found that the causal fungal agent enters the tree through pruning wounds. If this is so, in orchards where trees are not pruned or are pruned only slightly, very little sign of the disease should be present and this was precisely the case in one of the orchards visited in Somalia. Even though this gummosis-free, unpruned grapefruit orchard was only nine years old, many other orchards in the same area showed severe gummosis, and in all of these orchards pruning is carried out.
TABLE 14 How to distinguish between Rio Grande gummosis, scaly bark psorosis and phytophthora gummosis
| RGG | SBP1 | PG2 | |
| Gumming | Very copious (stalactites) | Inconspicuous | Copious |
| Site of gumming or scaling | All trunk and major branches | All trunk and major branches | Trunk at soil level |
| Part of bark affected | Outer layers | Outer layers | Entire bark |
| Gum pockets in wood | Present | Absent | Absent |
| Exposed wood at old lesions | Yes | No | Yes |
| Typical illustration | Figs 236 to 239 | Figs 98 to 100 | Figs 222 and 230 |
Notes:
1 Scaly bark psorosis.
2 Phytophthora gummosis.
Once infection invades the trunk it is practically impossible to eliminate the disease by surgery. Control has to be by prevention. Pruning should be reduced to a minimum and only small branches with a diameter not larger than 25 mm should be cut. Pruning cuts should be disinfected and, when dry, covered with wound dressing or asphalt. In California, carbolineum with 2 percent phenols has been recommended as a disinfectant for pruning wounds, and for final coating on the disinfected wood, low melting-point asphalt mixed with an equal quantity of carbolineum.
Heat- or frost-injured areas are also ports of entry of the RGG agent. Whitewashing of trunks and branches that might become exposed to sun (after heavy pruning) should be carried out with a zinc-copper-lime mix.
