Apiognomonia errabunda
(anthracnose)

A. errabunda and A. quercina are invasive species both in Europe and the USA for the following reasons: the pathogen has a three-way survival capability, with a conidial form on the twigs, leaves and buds of the tree; a sexual form with perithecia on fallen leaves, and an active mycelium on the leaves, buds and twigs in Mediterranean countries with mild winters; it produces a great number of conidia, 49,300/m³ air, at a distance of 10 m from the inoculum source after 10 days of sporulation, and 2400 conidia/m³ air at a distance of 1000 m from the source after 40 days of sporulation; it is highly adaptable to a wide variety of different climates in Europe, from parts of Russia through the countries of north-eastern Europe to the Mediterranean, and in North America, from Canada down to the Gulf of Mexico along the Pacific coast; and it has a very extensive host range.

Detailed information on the distribution of Discula and some other genera in Italy may be found in Anselmi et al. (2002).

A. errabunda and A. quercina are not subject to quarantine regulations because they are widespread. It would nevertheless be desirable to have such regulations for countries where the two pathogens do not occur, but where there are large populations of beech and oak.

The many associations between oaks and other trees in mixed stands have been described for central Europe by Mayer (1984) and Ellenberg (1988). In simple terms the prototype of such associations can be taken to be the mixed stand of oak and hornbeam (Carpinus betulus), in which Quercus petraea and/or Q. robur grow mixed with hornbeam in mesophile conditions on fertile hilly terrain. From these associations other important groups branch out, growing in different ecological conditions. On the more arid side there is the oak-birch association, with a high proportion of Q. petraea, though this species thrives less here. Where the soil is fresher and richer in nitrogen, the predominant association becomes one of maple and ash, with Q. petraea and/or Q. robur at a strong competitive disadvantage. On very humid soils the alder-elm combination comes to the fore and Q. robur can easily renew itself only if it is periodically flooded with alluvial silt (Perrin, 1954).

For the southern Mediterranean it is difficult to give a general picture of oak groves in a few words because of their diversity. In Italy, a reference oak stand can be taken to be one in which Q. pubescens and Q. cerris are mixed with hop hornbeam (Ostrya carpinifolia), which here replaces Carpinus betulus, though hornbeam remains the main competitor of the oaks. In more arid areas, the hop hornbeam becomes less successful and the only formations that remain are skimpy stands of Q. petraea and flowering ash (Fraxinus ornus), with an undergrowth of herbaceous species of dry brome grass (Ubaldi et al., 1984).

In more acidic soils the most successful stands are those containing Q. cerris, but also Q. petraea and chestnut (Oberdorfer and Hofmann, 1967). The latter species are replaced further south in Italy by Q. cerris with various species of heather (Erica) and Coronilla emerus, which are more thermophilous (Giordano and Schirone, 1989).

In hilly terrain in Italy it is not uncommon to see stands of Q. pubescens and Q. cerris with smaller admixtures of hop hornbeam. In the Po Valley there are plain-growing oak groves with Q. robur and Carpinus betulus, or with Q. robur, Q. cerris and Carpinus betulus (Agostini, 1965; Paiero, 1965). In coastal areas of central and southern Italy, the remaining plain-growing stands consist of Q. robur, Q. cerris, Q. frainetto (only in the south) as well as cork oak and Q. ilex (Corti, 1955; Padula, 1985).

The Apiognomonia complex includes numerous anamorph forms (apart from Discula umbrinella and D. quercina, which are dealt with in this datasheet), among which D. destructiva sp. nov. deserves a special mention for its wide distribution and pathogenicity. This species causes serious damage on Cornus florida and C. nuttallii in Canada and the USA (Redlin, 1991).

Information on the host range can be found in Farr et al. (1989), Kowalski and Kehr (1992), Phillips and Burdekin (1992), Toti et al. (1993), Wilson and Carroll (1994), Sahashi et al. (2000) and Anselmi et al. (2002).

An undefined species of Discula has been found in the leaves of Fagus crenata in Japan (Sahashi et al., 2000).

The biological and ecological characters given in this datasheet refer to A. errabunda sensu stricto and to A. quercina with their respective anamorphs, Discula umbrinella and Discula quercina.

A. errabunda and A. quercina are most easily found on Fagus sylvatica and Quercus spp., respectively; however, they have a vast host range to cover their geographic range. In North America they are found from the western provinces of Canada down along the coast to the Gulf of Mexico. In Europe they occur from central and southern Russia across north-eastern Europe, all the way to the countries of the Mediterranean. Such an extensive geographic range, with so many different climates, shows the adaptability of these fungi, though it is possible that ecotypes have differentiated within the species (A. errabunda and A. quercina) that are adapted to the particular climates of the two continents.

This is also seen in Italy, where A. quercina occurs in many different parts of the country, from areas with a semi-arid climate (e.g. Sicily, 37-38° N, 13-15° E) to those with a continental climate (e.g. Tuscany, 43-44° N, 10-12° E) (Anselmi et al., 2002).

In winter A. quercina survives mainly through its anamorph form (acervula) that differentiates on cankers along the branches and twigs. The teleomorph form (perithecia on dead leaves on the ground) ensures survival in more temperate areas.

The primary infection, in all areas, appears after the winter season when the temperature rises in the spring (18-20°C is sufficient for perithecia and acervula to mature). The ascospores and conidia then begin to infect the leaves, buds, shoots and twigs. The teleomorph Apiognomonia usually colonizes the leaves, buds and shoots, whereas the anamorph Discula colonizes not only the leaves and buds but also the twigs (bark and wood) (Ragazzi et al., 1999a). The ascospores and conidia are dispersed by wind, water and seeds, and possibly by insects as well (Petrini, 1991). Under test conditions, ascospores and conidia caused high levels of infection with the following regime parameters: day/night 16/8 h, 22/18°C, 40/65% RH and 20,000/0 lux (Ragazzi et al., 1999b).

The literature reveals that, on the whole, species of the Apiognomonia complex are adapted to temperatures ranging from 3 to 30°C (Neely and Himelick, 1967; Shishkina, 1969; Sinclair et al., 1987; Smith et al., 1988; Wilson and Carroll, 1994). The percent infected leaf area increases as the inoculum concentration increases from 100 to 100,000 conidia/ml, but then decreases at 1,000,000 conidia/ml. An inoculum concentration of 100,000 is therefore optimal for producing extensive leaf necrosis (Ragazzi et al., 1999a). Under experimental conditions of day/night 12/12 h, 25/15°C, 50/75% RH and 25,000/0 lux, conidia production gradually increased until it peaked at 23,000 conidia/m³ air, 14 days after sporulation. Conidia production is greatest between 21.00 h and 06.00 h (Ragazzi et al., 1999a).

The frequency of occurrence and extent of colonization are greater at lower altitudes and diminish as the altitude increases (Toti et al., 1993). Secondary infections are caused mainly by conidia that have differentiated in the canker lesions, produced by the primary inoculum, on the branches and twigs. Infected trees produce infected cones. The inoculum (conidia + ascospores) released from a mixed stand of Quercus cerris and Q. pubescens, averaging 15 m tall and growing at an altitude of 400 m a.s.l., decreases with distance from the inoculum source, so that 10 days after the start of sporulation, the number of propagules trapped in a spore trap increased from 49,300 propagules/m³ air at 10 m from the source to 14,200 propagules/m³ at 1000 m. After 40 days, the corresponding figures were 16,200 propagules/m³ at 10 m and 2400 propagules/m³ at 1000 m (Ragazzi, Università degli Studi di Firenze, Italy, unpublished data).

Individual species of the Apiognomonia complex are invasive in stands of Quercus spp. mixed with Fraxinus spp., Alnus spp., Castanea sativa, Fagus sylvatica, Corylus spp. and Acer spp, depending on location. In the USA, it is the pure oak stands that are most seriously affected. In both Europe and the USA, infection level is related to rainfall, increasing when rainfall is higher in the early part of the growing season (March-April in Europe and March-April-May in the USA). The incidence of A. errabunda on F. sylvatica increased on permanent observation plots in Alpine sites in Switzerland after the application of nitrogen fertilizer (Fluckiger et al., 1999).

A. errabunda sensu stricto is frequently found in association with Aureobasidium pullulans, Diplodina cf. microsperma, Epicoccum purpurescens, Microsphaeropsis sp., Phialophora sp., Phoma sp., Phomopsis sp. and Sporormiella intermedia.

A. quercina is associated with Alternaria alternata, Biscogniauxia mediterranea, Cladosporium cladosporioides, Cytospora sp., Diplodia mutila, Epicoccum spp., Monochaetia sp., Phoma cava, Phomopsis quercina, Trichoderma spp., Tubakia dryina and Ulocladium sp.

There are currently no known natural enemies of A. errabunda and A. quercina. However, it should be noted that on oak in Italy, the tree organs from which A. quercina is easily isolated are those that do not harbour any mycetes known to be biological antagonists of these agents. A. quercina is not found, or is much less common, at sites where potential antagonist species such as Acremonium, Aureobasidion, Paecilomyces, Ramichloridium and Trichoderma are frequent (Ragazzi, Università degli Studi di Firenze, Italy, unpublished data).

The acorn becomes infected, but it is not known where on the fruit the fungus is located, whether it contaminates the seeds, how long the fungus remains viable on the seed, or whether it can transmit the disease in this way.

A. errabunda (anamorph Discula umbrinella).

D. umbrinella can be isolated from leaves of Quercus alba and Q. rubra by the following method:

The fungus was isolated from leaf discs using the method of Wilson and Carroll (1994) with modifications in the solution concentrations and in the times and number of rinses during the sterilization procedure. A 6-mm-hole punch sterilized in 95% ethanol was used to remove six leaf discs located equidistantly along the midvein of each leaf. Each disc was placed on the sterile filter paper of a large vacuum filtration apparatus. The apparatus consisted of a plastic container (27.5 x 40 x 14.6 cm), an aluminium frame with an open aluminium lattice (33.1 x 46.2 x 2.8 cm), a sheet of porous Teflon filter (39.7 x 26.9 cm) (Reed Plastics, Rockville, Maryland, USA), and a sheet of sterile filter paper (39 x 26 cm) (Whatman, 3MM). The apparatus was attached in tandem to a vacuum flask and vacuum line. The entire apparatus was assembled within a laminar flow hood to maintain sterility throughout the isolation procedure. The sterile Whatman paper was placed on the Teflon surface of the vacuum filtration unit, premoistened with sterile distilled water, which was allowed to drag by gravity. The leaf discs were sterilized on the apparatus with 95% ethanol for 50 s, 70% ethanol for 50 s, 0.5% sodium hypochlorite with Tween 80 (0.2 ml/100 ml) for 2 min, and were then rinsed three times with sterile distilled water. Sterilization fluids were applied with squeeze bottles or pasteur pipettes. Excess liquid was vacuumed away from the leaf discs, which were then transferred to 24-well tissue-culture dishes containing 1 ml per well of oak leaf agar (OLA) consisting of 1.0 g dried and ground white oak leaves, 20 g Difco agar, 1000 ml distilled water. Prior to preparation of the OLA medium, leaves were collected, dried at 100°C for 15 min, shredded in a Waring blender for 1 min, then ground in a coffee grinder for 30 s. Dishes were incubated at 24°C under 12 h fluorescent lighting per day to induce conidiomata production.

Apiognomonia quercina (anamorph Discula quercina)

The isolation of D. quercina followed Ragazzi et al. (1999c). Two bark fragments of 3 cm² each were excised from Quercus cerris twigs, one from near the tip and one from near the base of the twigs, and two wood fragments were similarly removed, one from the base and one from the tip of the same twigs. Two fragments of embryo tissue and two of bud scale were also removed from each of two basal buds and two apical buds per twig. All fragments were sterilized by immersion in 3% sodium hypochlorite for 4 min followed by immersion in 60% ethanol for 2 min. The fragments were placed in Petri dishes, 9 cm in diameter, containing 20 ml PDA, plus 30 p.p.m. streptomycin to suppress bacterial growth. Incubation was in a controlled environment chamber at 22±2°C, 70% RH, with constant lighting at 15,000 lux.

Colony differentiation started after 4-5 days of incubation. Colonies were a very light brown, almost yellow, the mycelium was immersed, with hyaline, septate and branched hyphae. Growth, on oak leaf agar (OLA) at 25°C, was slow, reaching a diameter of ca 6.5 cm after 8 days. Acervula, 150 µm in diameter, epidermic, separate or confluent, light brown, smooth-walled and with an irregular dehiscence were produced after about 5 days on impoverished PDA (6 g/l potato dextrose broth (PDB) + 20 g/l agar). Conidiophores were 13-3-5 x 21 µm, hyaline, septate, single or branched (but only at the base), straight or slightly curved, elongated towards the tip. Conidia were 3.5-5 x 9-15 µm, hyaline, oblong, ellipsoid, not septate, with a smooth, thin wall, a blunt tip and the base more or less truncated.

PDA was certainly an excellent medium for the isolation of A. quercina, but for colony growth the best medium was OLA, pH 6.6, prepared by boiling 30 g oak leaves in 350 ml water for 30 min and adding 8 g agar (Difco). The best growth conditions in a controlled environment were: 30 W fluorescent lighting, a 12-h day with day/night temperatures 24/18°C; 50/75% RH, light intensity 15,000/0 lux. Under such conditions the colony differentiated in 3 days and reached a diameter of 9 cm in 6 days (Ragazzi et al., 2002). Other authors have carried out similar experiments, the most important of which have been Neely and Himelick (1967) and Wilson and Carroll (1994).

Neely and Himelick stated that isolates of Gnomonia quercina from white oak were studied on 10 agar media (Difco) with added bean pod, cabbage infusion, corn meal, lima bean, malt extract, potato dextrose, prune and Sabourand-dextrose. Growth rates were determined at temperatures ranging from 3 to 30°C.

Conidia production was greatest on bean pod, malt extract and PDA. No isolate produced many conidia on cabbage infusion, corn meal or Sabourand-dextrose. All isolates tended to form zonation rings on many of the media. The aerial mycelium of all isolates was white tending to pale grey on all media. Zonation rings varied in colour from grey to black. On PDA all isolates produced a dark-yellow pigment that spread throughout the medium after incubation for 4-6 weeks at 21°C. Colonies reached their greatest diameter size (7-9 cm) on prune, Sabourand-dextrose, corn meal and PDA. The optimal temperature for growth was 18°C.

Wilson and Carroll (1994) isolated D. quercina from various tissues of Quercus garryana. Surface sterilization of tree tissues was carried out as described in Wilson and Carroll (1994):

- Acorn shells (60 s in 95% ethanol, 60 s in 70% ethanol, 5 min in 33% NaOCl, 5 washes in sterile, distilled water)
- Naked seeds (shelled acorns) (30 s in 70% ethanol, 10 min in 10% NaOCl with 1% SDS, 5 washes in sterile, distilled water)
- Twigs, bark, wood (60 s in 95% ethanol, 2 min in 70% ethanol, 8 min in 33% NaOCl, 5 washes in sterile, distilled water)
- Leaves (50 s in 95% ethanol, 60 s in 70% ethanol, 5 min in 33% NaOCl, 4 washes in sterile, distilled water)
- Prebud burst and young postbud burst leaves (25 s in 95% ethanol, 30 s in 70% ethanol, 2 min in 33% NaOCl, 4 washes in sterile, distilled water).

Note:
NaOCl: dilutions (by volume) from a 5% sodium hypochlorite household bleach stock solution.
SDS: sodium dodecyl sulfate is a detergent used as a wetting agent.

Isolation was attempted from three organs:

Shelled acorns: the shell was removed, leaving only the cotyledons, which were attached at the embryo region. A single piece of each acorn seed (about a quarter of the total acorn) was surface sterilized. The pieces were placed in 22-mm-diam. sterile test tubes with 2 ml sterile water, and incubated under ambient laboratory conditions for 6 weeks, then scored for D. quercina.

Twigs: one 12-cm length of 1-2- and 3-year-old twigs was cut from trees. These were put on ice, taken back to the laboratory and immediately surface sterilized. The terminal 1 cm of twig length was cut off, then the remaining 10-cm length was cut into four equal sized parts. All 40 1-year-old segments, and 10 out of the 40 segments of the 2- and 3-year age classes of twigs were placed onto PDA and left to incubate under ambient laboratory conditions, then scored for the presence of the endophyte. All the remaining 2- and 3-year-old twig pieces had the bark removed from the wood. Bark of 2- and 3-year-old twigs can be prized off the wood with a sterile razor blade. The bark and wood were both separately placed onto PDA, incubated for 3 weeks under ambient laboratory conditions, then scored for the presence of the endophyte.

Increment cores of the tree trunk: two core samples per tree were taken at breast height from five trees. As oak trees are difficult to core with increment corers, the cores only went 10 cm into the tree trunk. To prevent contamination of the core sample with bark fungi, a small section of bark was removed from the tree around the point where the increment corer was inserted. The increment corer was sterilized in the field by flaming with ethanol. The cores were immediately placed in sterile glass tubes on ice, taken to the laboratory, surface-sterilized, broken in half, placed on PDA, incubated, and scored for D. quercina.

Studies to establish the cultural characteristics of A. quercina can be found in Stoneman (1898), Edgerton (1908), Westerdijk and van Luijk (1920) and Schuldt (1955).

A. errabunda and A. quercina cause a type of leaf spot that is diagnostic of these fungi: no other known biotic or abiotic agents produce similar spots. The presence of these two mycetes is practically confirmed when the spots are accompanied by cankers on the twigs, caused by the anamorphs Discula umbrinella and D. quercina, respectively. Absolute certainty can only be attained by isolating the fungi.

Toti et al. (1993) isolated D. umbrinella from the buds, twigs and leaves of Fagus sylvatica as follows: twigs from 10 randomly selected F. sylvatica trees were collected in March 1991 in Switzerland. One branch, 1-1.5 cm in diameter and carrying at least 30-40 buds, was excised from each tree. Five buds, along with the branch segment bearing the buds, were taken from each branch and surface sterilized by sequential immersion in 75% ethanol (1 min), NaOCl with 3-5% available chlorine (5 min), and 75% ethanol (30 sec). Each bud was cut longitudinally and the outer scales removed. The young, rolled-up leaf was cut into two parts, and the first 3-cm length of each piece of contiguous twig was sectioned longitudinally into two pieces. The twig pieces directly underneath the buds were contiguous pieces, as distinct from the distal pieces, which were separated from the buds by the contiguous twig pieces. Bud scales, rolled up leaf pieces, and twig pieces were placed in that order on 2% MEA (2% agar, 1% malt extract, Difco) supplemented with 50 p.p.m. terramycin to suppress bacterial growth. All plates were incubated at 20±2°C for up to 3 weeks, depending on the growth rate of the fungi. Isolation was by transfer of mycelium to MEA slants. Near-UV light (Philips TL 40W/05) was used to induce sporulation. Other detection methods are described in 'Diagnostic Methods'.

In addition to traditional detection methods, a number of biomolecular techniques are now widely available in plant pathology. Haemmerli et al. (1992) identified and compared isolates of D. umbrinella in F. sylvatica, Castanea sativa, Quercus petraea and Q. rubra using the following protocol:

Fungal isolates
All strains of D. umbrinella were isolated from symptomless leaf tissues of F. sylvatica, C. sativa, Q. petraea and Q. rubra as described by Sieber and Hugentobler (1987); all isolates were mycelial isolates. Cultures were maintained on 2% MEA slants (2% malt extract, 2% agar, both Difco ) at 4°C.

DNA isolation
Isolates were grown for 10 days at 20°C in shake culture containing 50 ml of V8 juice. The mycelium was removed by centrifugation (10 min at 3000 x g), lyophilized and ground to a fine powder. Mycelial DNA (30-40 mg) was extracted and purified by a total DNA CTAB mini-prep extraction method (Zolan and Pukkila, 1986).

PCR amplification conditions
Amplification reactions were carried out in volumes of 25 µl containing 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2 mM MgCl2 (for primers P1, P2, P14, OPE2, OPE4, OPE7, OPE12, OPE15); 2.5 mM MgCl2 (primers P11, OPE3, OPE5, OPE6, OPE11, OPE16, OPE20); 3 mM MgCl2 (P4, OPE1); 100 µM each of dATP, dCTP, dGTP and TTP (Boehringer Mannheim); 0.2 M primer; 25 ng of DNA; and 1 U of Taq DNA polymerase (Boehringer Mannheim). Amplification was performed in a Perkin Elmer Cetus Gene Amp PCR system 9600 programmed for two cycles of 30 sec at 94°C, 30 s at 36°C, 120 sec at 72°C, 36 cycles of 20 s at 94°C, 15 s at 36°C, 15 s at 45°C, 90 s at 72°C, followed by 10 min at 72°C. Reaction products were resolved by electrophoresis (4 V/cm) in a 1.5% agarose gel, run in 1 x TPE for 4 h and stained with ethidium bromide.

A total of 17 random primers, each with a length of 10 nucleotides and a minimum GC content of 50% were used. The primers were supplied by Operon Technologies Inc., Alameda, California, USA, and some were synthesized by MicroSynth AG, Windisch, Switzerland.

Hybridization and autoradiography
Three RAPD marker products: no. 211, a 2100-bp product of isolate 11 by primer P2; no. 214, a 1170-bp product of isolate 2 by primer P14; and no. 1814, a 1000-bp product of isolate 18 by primer P14, were transferred from the agarose gel to reaction tubes using autoclaved toothpicks and amplified under the conditions described above. Twenty-five nanograms of reamplified DNA was labelled with 50 µCi (a-32P)dCTP (3000 Ci/mmole; Amersham International; random primed DNA labelling kit, Boehringer Mannheim) and used as hybridization probes on Southern blots. EcoRI-digested total genomic DNA was separated by horizontal agarose electrophoresis for 15 h and blotted to Hybond N+ membranes (Amersham) (Southern, 1975). Prehybridization and hybridization reactions were carried out in plastic bags at 65°C with 5 x SSPE, 5 x Denhardt's solution, 1% SDS, 0.5 mg of herring sperm DNA. Hybridizations were incubated at 65°C for 14 h.

Data analysis
The occurrence of each amplification product in the gel was scored as 1, non-occurrence as 0. The resulting matrix was used to compute indices of between-isolate distance using the PCT option offered by the software package SYSTAT 5.1 (Wilkinson, 1989). This matrix computes the percentage of value comparisons resulting in disagreements in two column or row profiles, and is particularly useful for categorical or nominal scales as it does not apply any weighting to the matrix. The dendrogram was constructed by UPGMA-clustering (Sneath and Sokal, 1973).

Due to the variable regulations around (de)registration of pesticides, your national list of registered pesticides or relevant authority should be consulted to determine which products are legally allowed for use in your country when considering chemical control. Pesticides should always be used in a lawful manner, consistent with the product's label.

Any methods of control must be applied to trees in the nursery or to young trees in the field. On adult trees, the general decline caused by A. errabunda on beech and A. quercina on oak, involves other organisms and is therefore difficult to control.

In the nursery and arboretum it is good practice to gather up the leaves, twigs and branches that fall to the ground, to keep the trees in good condition by proper watering and fertilizing, and to ensure good ventilation.

Although it remains doubtful whether A. quercina is transmitted by seed, it is nevertheless strongly advisable to use healthy seed (checked to be free of the mycete) or seed from healthy trees. Any beech or oak trees that become infected in adult stands must be eliminated.

Chemical treatments are rare. Only in cases where anthracnose has defoliated oak trees for 3-5 years in succession is thiophanate-methyl applied to start vegetative growth, with repeated treatment after 7-10 days. Additional spraying may be carried out when the growing season is cool and humid (see http://www.entomology-umn.edu/cues/dx/CB/oanthab.htm, the site of the Center for Urban Ecology and Sustainability, The University of Minnesota, USA).

One promising means of control is to promote fungi that are biological antagonists against A. quercina and occur on the same organs on the oak tree that yield the disease agent.