Cylindrocarpon destructans var. destructans AND Neonectria discophora var. rubi ASSOCIATED WITH BLACK FOOT ROT ON BLACKBERRY(Rubus glaucus Benth.) IN MÉRIDA, VENEZUELA

Luis Cedeño, Chrystian Carrero, Kleyra Quintero, Henry Pino and Wilmer Espinoza

Luis Cedeño. Agronomist, Universidad de Oriente, Venezuela. Master in Plant Pathology, University of Georgia, USA. Researcher, Laboratory of Phytopathology, Instituto de Investigaciones Agropecuarias (IIAP), Universidad de Los Andes (ULA), Mérida, Venezuela. Address: IIAP-ULA. P.O. Box. 77 (La Hechicera), Mérida 5101-A, Venezuela. e-mail: cedenol@ula.ve

Chrystian Carrero. Agronomist, Universidad del Zulia, Venezuela. Master in Forest Management, ULA. Researcher, Laboratory of Phytopathology, IIAP-ULA.

Kleyra Quintero. Phytotechnician, Assistant, Laboratory of Phytopathology, IIAP-ULA.

Henry Pino. Phytotechnician, Fruit Trees Section, IIAP-ULA.

Wilmer Espinoza. Agricultural Technician, General Agronomy Section, IIAP-ULA.

Resumen

En una plantación comercial de mora (Rubus glaucus Benth.) ubicada en El Valle, Municipio Libertador, estado Mérida, Venezuela, en 1999 se detectó una enfermedad de pudrición negra del pie. El agente causal fue identificado como Cylindrocarpon destructans var. destructans (teleomorfo= Neonectria radicicola var. radicicola). Este patógeno ataca las raíces y la corona, induciendo muerte regresiva en las cañas. Síntomas similares a los observados en el campo fueron reproducidos en plántulas de mora cultivadas en suelo inoculado (1,5%, p/p) con granos de cebada colonizados por el hongo. Las plántulas control no desarrollaron síntomas de enfermedad. C. destructans var. destructans fue aislado consistentemente de las plántulas crecidas en suelo contaminado. En enero y mayo 2002, en áreas de Miraflores (Municipio Campo Elías) y Tabay (Santos Marquina), respectivamente, se observaron daños similares, pero con la diferencia que las plantas moribundas mostraron lesiones de color violeta en los tejidos vasculares de la porción basal de las cañas, mientras las plantas muertas tenían abundantes peritecios de color rojo en el cuello, la corona y las raíces. En diciembre 2003, los mismos síntomas y signos fueron detectados en plantas muertas en Santa Rosa (Municipio Libertador). El microorganismo comúnmente asociado con esos síntomas fue identificado como Neonectria discophora var. rubi, fase sexual de Cylindrocarpon ianthothele var. ianthothele. Estes es el primer reporte documentado de C. destructans var. destructans como causa de una enfermedad de pudrición negra del pie en R. glaucus y es también el primer reporte de N. discophora var. rubi en una especie Rubus en Sur América.

Summary

In a commercial blackberry (Rubus glaucus Benth.) field located at El Valle, Municipality Libertador, Mérida State, Venezuela, a black foot rot disease was detected in 1999. The causal agent was identified as Cylindrocarpon destructans var. destructans (teleomorph=Neonectria radicicola var. radicicola). This pathogen attacks the roots and the crown, inducing die-back in canes. Symptoms similar to those observed in the field were reproduced on blackberry seedlings grown in soil inoculated (1.5% w/w) with barley grains colonized by the fungus. Control seedlings did not develop disease symptoms. C. destructans var. destructans was consistently isolated from the seedlings growing in contaminated soil. In January and May 2002, in areas of Miraflores (Municipality Campo Elías) and Tabay (Municipality Santos Marquina), respectively, similar damages were observed, but with the difference that the dying plants showed violet lesions on the vascular tissues in the lower parts of the canes, while the dead plants had abundant red perithecia on the neck, crown and roots. In December 2003, the same symptoms and signs were detected on dead plants at Santa Rosa (Municipality Libertador). The microorganism commonly associated with these symptoms was identified as Neonectria discophora var. rubi, sexual stage of Cylindrocarpon ianthothele var. ianthothele. This is the first documented report of C. destructans var. destructans as a cause of black foot rot disease on R. glaucus, and it is also the first report of N. discophora var. rubi on a Rubus species in South America.

Resumo

Em uma plantação comercial de amora (Rubus glaucus Benth.) situada em El Valle, Município Libertador, estado Mérida, Venezuela, em 1999 detectou-se uma enfermidade de podridão negra do pé. O agente causal foi identificado como Cylindrocarpon destructans var. destructans (teleomorfo= Neonectria radicicola var. radicicola). Este patógeno ataca as raízes e a coroa, induzindo morte regressiva na cana. Sintomas similares aos observados no campo, foram reproduzidos em plântulas de amora cultivadas no solo inoculado (1,5%, p/p) com grãos de cevada colonizados pelo fungo. As plântulas controle não desenvolveram sintomas de enfermidade. C. destructans var. destructans foi isolado consistentemente das plântulas crescidas em solo contaminado. Em janeiro e maio 2002, em áreas de Miraflores (Município Campo Elías) e Tabay (Santos Marquina), respectivamente, observaram-se danos similares, mas com a diferença que as plantas moribundas mostraram lesões de cor violeta nos tecidos vasculares da porção basal da cana, enquanto as plantas mortas tinham abundantes peritécios de cor vermelha no caule, na coroa e nas raízes. Em Dezembro 2003, os mesmos sintomas e sinais foram detectados em plantas mortas em Santa Rosa (Município Libertador). O microorganismo comumente associado com esses sintomas foi identificado como Neonectria discophora var. rubi, fase sexual de Cylindrocarpon ianthothele var. ianthothele. Este é o primeiro relatório documentado de C. destructans var. destructans como causa de uma enfermidade de podridão negra do pé em R. glaucus e é também o primeiro relatório de N. discophora var. rubi em uma espécie Rubus na América do Sul.

KEYWORDS / Black foot rot / Blackberry / Cylindrocarpon / Neonectria /

Received: 04/21/2004. Modified: 07/02/2004. Accepted: 07/16/2004.

Introduction

In Venezuela blackberry (Rubus glaucus Benth.) production takes place in the Aragua, Barinas, Lara, Mérida, Táchira and Trujillo States. Although in the context of the national agricultural system this minor fruit-bearing species does not have an outstanding economic and social importance, in the Andean region, particularly in the Mérida State, it is popular and represents an important primary source of revenue for small and medium size producers. Their fruits are consumed fresh and processed in diverse ways as syrup, ice creams, marmalades, juice, wines and yogurt.

The main natural problems of this crop in the Venezuelan Andes are the diseases caused by the fungi Glomerella cingulata (Stoneman) Spaulding. & H. Schrenk, (anthracnose; Cedeño and Palacios, 1991), Sphaerotheca macularis (Wallr.: Fr.) Lind, (powdery mildew; Cedeño et al., 1995a), Peronospora sparsa Berk. (downy mildew; Cedeño et al., 1995b), Coniothyrium fuckelii Sacc. (cane blight; Cedeño and Carrero, 2000) and Botrytis cinerea Pers.: Fr. (gray mold), which reduce the yields, alter the quality of the fruit and diminish the productive life of the plants. Because of the high incidence of these pathogens, the current amount of blackberry in the regional market does not satisfy the demand; moreover, the fruit is of very low quality and tends to spoil quickly.

Without concerted efforts on the part of the Autonomous Agricultural Health Service (SASA) and growers to remove and destroy all infected materials in order to avoid or diminish the incidence and/or dissemination of the pathogens, the future of blackberry production in these regions is in doubt.

In May 1999, a disease of unknown cause was discovered in a commercial field of blackberry located in the village of Monterrey at El Valle, Municipality Libertador, Mérida State. Plants were affected by a die-back disease. The dying plants showed symptoms of wilt, and the dead ones had lost all their leaves. In some leaves of the dying plants necrosis was visible only at the tip and the borders, while others were completely dry and brittle; all the leaves were strongly curled toward the upper surface.

The dying floricanes had mummified fruits (dry and hard), in addition to apparently unaffected mature and immature fruits. Some mummified fruits were greenish brown, while others were dark brown to almost black. The roots and the crown of the dead plants were affected by a black rot (Figure 1). Transverse cracks were seen in the roots, and in them the cortex came off with ease, revealing light brown, dark brown and black streaks in the vascular tissues. In the internal portion of the crown of dying plants, black streaks were also seen, which contrasted with the yellowish white color of the surrounding healthy tissues. All the dead plants had a hollow central portion at the crown. Later, similar symptoms were detected in plants grown in sectors of Macho Capaz (Municipality Campo Elias) and La Azulita (Municipality Andrés Bello).

In February and May 2002, on roots and crowns of plants dead by black foot rot at Miraflores and Tabay, respectively, clusters of reddish to reddish brown perithecia were commonly found (Figure 2). The anatomical and morphological features of perithecia were characteristic of members of the Order Hypocreales, Subdivision Ascomycotina (Gerlach and Nilssen, 1963; Rossman, 1983; Samuels and Brayford, 1990; Rossman et al., 1999; Brayford et al., 2004). In Tabay, it was common to observe a violet pigmentation on the basal portion of dying canes (Figure 3), while the roots and the crown of the dead plants showed black rot symptoms.

The present work was carried out with the purpose of establishing the identity of the microorganisms causing the described symptoms, and to evaluate their pathogenicity.

Materials and Methods

Isolation and identification of the fungal pathogens

Isolations were made from symptomatic roots and crowns collected in sectors of El Arado, La Azulita, Macho Capaz, Miraflores, Monterrey and Tabay, all located in Mérida State. After washing the material with running tap water for 1h, small pieces (ca. 2-3mm) were taken from the interface of healthy and diseased tissues; these immediately were surface sterilized with 0.5% sodium hypochlorite (NaOCl) for 3min, rinsed three times in sterile distilled water (SDW), dried on sterile absorbent paper and then plated onto Petri dishes containing water agar acidified (pH 4.5) with lactic acid (AWA). The dishes were incubated at 25 ±1ºC in the dark and, later, the emergent colonies were transferred to test tubes containing slants of potato-dextrose agar (PDA; Difco). Additionally, perithecia that had been briefly surface disinfested with 0.5 NaOCl, were placed directly on AWA.

Following the procedure of Hansen and Smith (1932), 10 monoconidial and 10 monoascosporic cultures were produced, respectively, using one of the numerous mass isolates obtained from infected roots and crowns collected in the sectors where perithecia were not found, and from ascospores exuded by perithecia developed on PDA. The parameters evaluated in the monoconidial culture were the morphology of the colonies on PDA and the form and size of the asexual reproductive structures (microconidia, macroconidia and chlamydospores) produced in vitro, while in the monoascosporic cultures they were the morphology of the colonies on PDA, the form and size of the asexual structures (macroconidia) developed in vitro, and those of the sexual structures (perithecia and ascospores) formed in situ and in vitro. In both cases, the cultures were incubated at room temperature (22ºC) and 12h of light from a lamp placed at 45cm (2 tubes of fluorescent daylight F40D Extralife, Former 40W, and 2 tubes of black light Roblan 110V, BLB 40W).

The identification was carried out comparing the information registered with those published in the specialized literature (Booth, 1966, 1967; Samuels and Brayford, 1990; Brayford et al., 2004). Fifty microconidia, macroconidia or chlamydospores were measured in each monoconidial culture, while in each monoascosporic isolate 50 macroconidia, 25 perithecia, and 50 ascospores were measured.

Growth rate

The selected cultures were grown in plates containing 20ml of potato-sucrose agar (PSA; Booth, 1966), cornmeal agar (CMA; Difco) or PDA, which were inoculated with disks (6mm diam) taken from 10-days-old colonies on 1.2% water agar medium. Five plates of each substratum were used and they were incubated for 7d at 22ºC in the darkness. The radial growth (mm/day) of the mycelium was calculated measuring in each colony 2 perpendicular diameters, substracting the diameter of the initial inoculum and dividing the result by two.

Pathogenicity tests

For the inoculation tests with the monoconidial culture, blackberry seedlings 6-8cm tall were used. They were grown in black polyethylene bags containing soil sterilized for 1h with dry heat at 110ºC during 3 successive days. Before inoculation the seedlines were placed in a human chamber with the purpose of preventing stress resulting from transplanting. The inoculum was produced by growing the fungus in test tubes (25x150mm) containing barley grains, prepared as follows. A layer (2-3cm) of moistened sterile cotton was placed in the bottom of each tube and barley grains were placed on top of the cotton to a height of 2cm. The tubes were sealed with cotton protected with gauze and sterilized twice, with an interval of 24h, for 20min at 121ºC and 15lb·in^-2. Each tube was inoculated with four disks (6mm diam) of mycelium taken from a 5-days-old culture grown on PDA. Cultures were incubated at room temperature (22ºC) and under 12:12h (light:dark) regime for 30d. The inoculum was applied at 1.5% (w/w) and each seedling received a 1:50 dilution in SDW. Seedlings used as control only received a suspension of barley grains not colonized by the fungus. The inoculum was covered with soil and the seedlings were transferred to the greenhouse where they remained covered with bags of transparent plastic for 3d. Starting 3d after inoculation (dai), the seedlings were examined periodically to evaluate the development of foliage symptoms. Regularly, some seedlings were examined to observe damages on the roots and on the neck.

The pathogenicity tests of the monoascosporic cultures were done in 9cm disposable Petri dishes (Unestam and Stenström, 1989) containing a substratum made of granulated vermiculite, dolomitic lime and peat (VT-M Premier Sogemix, Vegetable Transplant Growing Mix, pH 7.0), which was sterilized for 1h at 121ºC and 15lb·in^-2 during 3 consecutive days. The roots of 3½ months old seedlings were placed on the substratum and the roots carefully separated, while the foliage came out through a circular hole that included the cover and the base of the dish. Ten seedlings were inoculated by wounding and ten with no wounds, applying to each one 5ml of a monoconidial suspension (1.62 macroconidia/ml) on the root system. As a control 3 wounded seedlings and 3 unwounded seedlings were used and SDW was applied on them. The dishes were sealed with a double layer of Parafilm® perforated with a dissection needle to allow aeration, covered with aluminum foil to prevent illumination of the roots, and finally placed in vertical position during 3d under the lamp (12:12h light/dark regime). Later, the seedlings were transferred to the greenhouse, where they were watered twice per week with a nutrient solution (potassium nitrate 0.5g; Epsom salts 0.5g; di-Ammonium phosphate 0.16g; calcium nitrate 0.89g; water up to 1000ml).

All the inoculation tests were carried out twice. Isolations were made from the seedlings infected experimentally to prove the Koch postulates.

Results

Isolation and identification of fungal pathogens

Only two fungi were isolated from infected crowns and roots of R. glaucus, and from perithecia. Based on the morphology of the asexual reproductive structures produced in situ and in vitro, they were recognized as different species of Cylindrocarpon Wollenw. (Booth, 1966).

One of the fungi isolated consistently formed microconidia, macroconidia, and chlamydospores. It was obtained from all the materials coming from the different geographical locations sampled, except from Miraflores and Tabay. Based on the type, morphology and size of asexual structures produced in vitro, the fungus was identified as C. destructans var. destructans (Zinssm.) Scholten [=C. radicicola Wr.] (Booth, 1966; Seifert and Axelrood, 1998), whose teleomorph, Neonectria radicicola (Gerlach & Nilsson; Mantiri & Samuels [=Nectria radicicola Gerlach & Nilsson] (Gerlach and Nilsson, 1963; Booth, 1967; Samuels and Brayford, 1990; Mantiri et al., 2001) was discovered in Sweden on rotten leaves, peduncles and bulbs of Cyclamen persicum L. (Gerlach and Nilsson, 1963).

On PSA, HMA and PDA media, the mycelial growth of C. destructans var. destructans, averaged 3.2, 2.6 and 2.3 mm/day, respectively. On PDA the colonies produced cottony aerial mycelium that was grayish-white at first, later becoming cream-colored to light brown. From the reverse, the cultures were reddish-brown in the central portion and beige in the borders. When the dishes were opened a distinctive odor of musty earth was detected.

The microconidiophores develop as lateral phialides or terminally on short lateral branches which also may form one or more laterals, each terminating in one or more cylindrical to awl-shaped phialides. The phialides measured 18.0-35.0 x 2.5-3.0µm. The microconidia were hyaline, cylindrical to oval and 7.2 (5.6-8.8µm) x 3.9 (3.3-4.5µm). The macroconidiophores are produced as lateral branches with elongated stipe and loosely branched apex, each branch terminating in one or more phialides. The macroconidia were hyaline, cylindrical or slightly wider at the distal end, straight or curved with rounded ends and a protuberant basal scar, 1-3 septate, although occasionally some with 4 or 5 septa were observed. The 1-septate macroconidia measured 26.1 (23.1-29.2µm) x 5.8 (5.2-6.2µm) and the 2-3 septate measured 37.4 (34.5-40.3µm) x 7.4 (6.8-8.0µm). The 5-septate macroconidia averaged 37.0 (36.0-38.0µm) x 7.0µm. The chlamydospores appeared on and inside the substratum forming terminal and intercalary chains and they were spherical to elliptic in shape, hyaline at the beginning and then with thick cellular golden brown walls, 12.9 (11.3-14.5µm) x 11.4 (10.1-12.7µm) in diameter, smooth, although deposits of substances that made them appear rough were frequently observed on the surface.

The teleomorph of C. destructans was not found in situ and was not produced in vitro, which suggests that it is a heterothallic species.

The second Cylindrocarpon fungus was isolated from roots, crowns and perithecia taken from diseased plants sampled in sectors of Miraflores and Tabay. The cultures originating from perithecia always formed perithecia on PDA. Clusters of perithecia were found on roots with black rot (Miraflores) and on the neck of canes with die-back (Tabay). Perithecia were ovoid to spherical, reddish to reddish-brown, 380-700 x 350-650µm, smooth and shining, with a slightly protruding, domed and darkened apical ostiole through which ascospore were extruded in white or buff tendrils. The perithecial wall did not show a pseudoparenchymatous structure but was composed of an intertwining network of thickened hyphae. Within the perithecia paraphyses and hyaline, cylindrical asci containing 8 ascospores in uniseriate disposition, were also observed. The ascospores were hyaline, ellipsoid to subfusoid with a simple septum, 11.6 (11.0-13.0µm) x 5.0µm, flat when young, but faintly spinulose and pale brown at maturity.

After 6 weeks of growth on PDA, all the monoascosporic cultures had produced fertile perithecia similar to those found in the field. The perithecia developed mainly in clusters that, in general, shared the same stromatic base and they showed the same transition in color from white, pale yellow, bright red, dark red, amber to finally almost black as seen in nature. The ascospores were extruded in creamy white colored masses deposited around the ostiole. The anatomical and morphological characteristics, as well as the size of the perithecia and ascospores that it contained, coincided with those described for Neonectria discophora (Mont.) Mantiri & Samuels [=Nectria mammoidea W. Phillips & Plowr. var. rubi (Osterw.) Weese] (Brayford et al., 2004), teleomorph of C. ianthothele var. ianthothele Wollenw., which differs from C. destructans in not producing microconidia or chlamydospores (Booth, 1966). The identity of the species was confirmed by Gary J. Samuels (Personal Communication).

On PSA, HMA and PDA, the mycelial growth of the selected monoascosporic cultures averaged 1.3, 0.5 and 0.6mm/day, respectively. On PDA the colonies showed a distinctive violet pigmentation similar to that observed on the neck of the canes infected naturally in Tabay. The colonies were floccose and the surface mycelium was violet while the aerial mycelium was yellowish brown; the colony reverse was dark.

The monoascosporic cultures also developed abundant macroconidia on violet-colored sporodochia, but no microconidia or chlamydospores, and strands of golden to dark brown hyphae. The conidiogenous cells were cylindrical, hyaline, with a collarette and thickened ring in the apex, and arose at the tips of short, much branched conidiophores. The macroconidia were hyaline, curved, cylindrical with round ends, 0-5 septate. Almost 90% of the conidia were 3-5 septate, being the 4-septate the most abundant. The 3-, 4- and 5- septate macroconidia averaged 52.6x6.8, 64.8x7.1 and 71.9x7.3µm, respectively.

The absence of microconidia and chlamydospores, together with the morphology and size of the macroconidia and violet color of the colonies confirm the identity of this species as C. ianthothele var. ianthothele Wollenw., and Neo. discophora (Booth, 1966, Mantiri et al., 2001, Brayford et al., 2004).

Pathogenicity tests

The inoculation tests with C. destructans var. destructans resulted in the production on seedlings with the same symptoms as observed in the field. When the plastic bags were removed, all the seedlings growing in contaminated soil had leaflets showing wilt symptoms and tip necrosis. The leaflets had lost their natural green color and showed the tendency to curl toward the upper surface. The necrosis began at the tip and at the borders, at first light brown and later becoming dark brown. Lightly sunken light brown, dark brown, violet, purple to black lesions were observed on the roots. The leaflets of the youngest leaves were the first to die. Some roots were completely blackened, collapsed and had lost the cortex. Of the seedlings, 60% died at 7dai and the rest died during the following two weeks. Seedlings inoculated with the control were unaffected. C. destructans var. destructans was the only microorganism consistently isolated from the seedlings experimentally infected, which demonstrates that it is the cause of black rot on the roots and crown of blackberry, R. glaucus.

The pathogenicity tests with Neo. discophora var. rubi were also successful. Five days after inoculation, in 70% and 40% of the seedlings inoculated with and without wounding, respectively, the younger leaves had died and other leaves showed tip necrosis and tended to curl towards the upper surface. The foliage of the control seedlings was not affected. One week later, 70% and 60% of the seedlings with and without wounding, respectively, had severe foliage damage, while the control seedlings remained unaffected. Five and one-half weeks after inoculation, 50% of the wounded seedlings and 50% of those without wounds were dead, while the control seedlings remained healthy. The roots of the dying seedlings showed the same violet color as was observed on the neck of canes affected by die-back in Tabay. Around the neck only few roots turned violet. The affected roots presented the following sequence of colors: violet, light brown, dark brown and black. The roots of dead seedlings had collapsed and blackened, and C. ianthothele var. ianthothele was frequently isolated from the roots artificially infected.

Discussion

The fungi species that formerly were considered in the genus Nectria are now located in the families Nectriaceae and Bionectriaceae (Rossman et al., 1999). Nectria and Neonectria are included in the Nectriaceae (Rossman et al., 1999, Mantiri et al., 2001). Species of Nectria s. str. include secondary pathogens of trees; they have red, anatomically distinctive perithecia and anamorphs in the genus Tubercularia. The species of Neonectria are easily distinguished from those of Nectria because of their Cylindrocarpon anamorphs; their perithecia are anatomically and morphologically different.

On the basis of analyses of mitochondrial ribosomal DNA sequences (Mantiri et al., 2001; Brayford et al., 2004), the species of Nectria with Cylindrocarpon anamorphs were placed in the genus Neonectria, including Neonectria discophora and Neonectria radicicola. Previously, Nectria radicicola had been included in the Nectria radicicola-group (Samuels and Brayford, 1990) and Nectria was included in the Nectria mammoidea-group (Booth, 1959, 1966; Rossman, 1983; Brayford et al., 2004). Each of these groups was characterized by perithecial anatomy and by anamorphs.

Booth (1966) recognized 27 species of Cylindrocarpon and distributed them among four groups, each distinguished by the presence or absence of microconidia and/or chlamydospores. The species included in the first group produce abundant microconidia but do not have mycelial chlamydospores. In the second group were placed those species that do not form microconidia nor chlamydospores. Those of the third group develop microconidia and chlamydospores. Those of the fourth group have chlamydospores but do not produce microconidia. Booth (1966) concluded that the name C. radicicola was antedated by C. destructans and located it in group 3; while he placed in group 2 the anamorphs of Neo. discophora var. discophora (=N. mammoidea) and Neo. discophora var. rubi (=N. mammoidea var. rubi). The var. rubi was retained as a variety of Neo. discophora because it has only been found on plants of the genus Rubus (Brayford et al., 2004); however, the tendency in var. rubi is to produce ascospores in culture and macroconidia that are somewhat shorter and wider than those of the var. discophora, and to grow slower in vitro. Whether var. discophora and var. rubi can be separated at the species level will require a more intense comparative study using DNA sequences.

The only species of Cylindrocarpon that has been associated with damage (canker and die-back) on Rubus plants grown in Europe (Brayford, 1991) is C. ianthothele var. ianthothele whose teleomorph, Neo. discophora var. rubi, was discovered for the first time in Switzerland on rotten roots of raspberry, R. ideaus, (Osterwalder, 1911). Later, this microorganism was found in association with similar damage on raspberry in Scotland (Alcock, 1925) and England (Nattrass, 1927; Pethybridge, 1927). However, pathogenicity of the fungus has not been demonstrated experimentally (Alcock, 1925; Nattrass, 1927; Pethybridge, 1927) and because of that it has been considered as a secondary pathogen of stressed plants (Brayford, 1991).

C. destructans is a cosmopolitan natural inhabitant of the soil, commonly associated with roots and residues of a wide variety of woody and herbaceous plants (Booth, 1966, 1967; Kluge, 1966; Samuels and Brayford, 1990), especially in alkaline soils and, less frequently, in soils of coniferous forests (Matturi and Stentöm, 1964). This fungus is considered to be a necrotrophic and opportunistic pathogen, because it only expresses its pathogenicity on plants subjected to stress or when conditions favor its development (Unestam et al., 1989; Brayford, 1991). However, in this study, the selected monoconidial strain of the fungus infected and rotted the roots of healthy, unstressed seedlings, indicating that it is a true pathogen of R. glaucus.

C. destructans is very sensitive to antagonism and to competition on the roots and because of that, in order for it to be able to compete successfully, it has to invade and become dominant in the weakened roots before the arrival of saprophytes (Unestam et al., 1989). In normal and biologically balanced soil, C. destructans does not become pathogenic (Kluge, 1966), but when the microbial community of the soil is altered by treatment with water steam or fungicides, toxins produced by the fungus might accumulate at toxic levels that favor the infection of the host plants. The severity of the attack by C. destructans on Pinus sylvestris L. is increased when the continuous use of fungicides inhibits the action of the antagonists (Unestam et al., 1989). C. destructans is a "pioneer colonizer" of roots because it has the capacity to grow and develop quickly in atmospheres with low O2 levels that are not favorable for other fungi (Ludeking and Relab den Haan, 2002).

The pathogenicity of C. destructans is related to the production of a toxin that weakens and kills the root tissues of plants affected by stress caused by transplant, root prune, anaerobiosis around the roots, shade produced by high plantation density and inappropriate use of certain pesticides (Kluge, 1966; Evans et al., 1967; Petäistö, 1982; Unestam and Stenström, 1989; Unestam et al., 1989; Beyer-Ericson et al., 1991). The stress caused by O₂ deficiency due to an excess of water in the soil (Unestam et al., 1989), or by the continuous use of fungicides (Unestam et al., 1989; Beyer-Ericson et al., 1991), weaken the root system facilitating the infection by C. destructans. Apparently, the soil reaction is not a critical factor because the fungus grows substantially at pH between 2.9 and 7.9 (Unestam et al., 1989). The toxin seems to have antibiotic effects on other fungi (Kluge, 1966; Unestam et al., 1989) because it inhibits the growth of saprophytes on infected roots and, also, it seems it has antibiotic activity in vitro against Trichoderma viride and other fungi (Kluge, 1966).

C. destructans possesses strains that differ widely in pathogenicity (Kluge, 1966). There is an intimate correlation between the production of toxin and the degree of virulence (Kluge, 1966; Unestam et al., 1989), and between virulence and the production in vitro of a dark-colored pigment (Unestam et al., 1989). The correlation between toxin production and pathogenicity indicates that the substance, probably of phenolic nature, plays an important role in the development of the root rot that affects the seedlings of P. sylvestris (Kluge, 1966). According to Ahn and Lee (2001), the virulence of the C. destructans that attacks ginseng (Panax quinquefolius L.), is controlled by a double helix viral RNA.

The pathogenicity tests evidenced that both of the isolated fungi are pathogens of R. glaucus, but Neo. radicicola was more aggressive, as it killed more seedlings in a shorter time. This difference in pathogenicity could be related to the rate of growth, since on PSA, PDA and CMA, Neo. radicicola grew 2.5, 3.8 and 5.2 times faster than Neo. discophora.

The growth rate of Neo. discophora was very low in all the substrata used, as compared to that of Neo. radicicola. The disease caused by Neo. discophora, thus, progresses more slowly than that induced by Neo. radicicola and, consequently, it requires that the conditions that facilitate and favor their occurrence converge and remain in place for longer periods. In this respect it is important to point out that the symptoms and signs of the disease caused by Neo. discophora, in general, have only been observed during later lapses to very humid years (Nattrass, 1927; Pethybridge, 1927) or very dry years (Brayford, 1991). In 1988 a severe attack of Neo. discophora occurred on the red raspberry of Scotland (Brayford, 1991), after a previous year whose severe summer compacted soil around the primocanes that damaged the tissues at the point of attachment to the crown. In Tabay, Mérida, Venezuela, a region where the fungus appeared associated with 11% of dead plants, the soil remained very humid for a long time; while in Miraflores the disease was observed after three months without rain. In December 2003, the disease was seen in adult plants that died in the Institute of Agricultural Research of Universidad de Los Andes, located in Santa Rosa, Mérida, after several months of abundant precipitation. It is possible that the pathogenicity of this microorganism is related to an excess of humidity after damages caused by the wind followed by water logging (Brayford, 1991) or with stress caused by long lapses of deficiency or abundance of water in the soil. It is our opinion that in the sites under study, the infection takes place through the wounds caused by pruning since they are not commonly treated for healing nor are they protected with fungicides. The slow development of the infection induced by this fungus could explain why Neo. discophora has always been found associated with old blackberry plants, while Neo. radicicola has been isolated from seedlings and from young and adult plants.

This is apparently the first report of C. destructans var. destructans as a cause of black rot on the roots and the crown in a species of Rubus. However, it is important to point out that perithecia of Neo. radicicola var. radicicola occur in native and disturbed vegetation in Venezuela. Samuels and Brayford (1990) reported specimens collected in 1971 in the mountains of Nirgua (Carabobo State, Dumont-VE 1525, on unknown vine), and in La Carbonera (Mérida State; Dumont-VE 2512, non identified wood). These specimens are preserved in the cryptogamic herbarium of the New York Botanical Garden. The literature related to the fungi that attack Rubus grown in America (Farr et al., 1989; Alfieri et al., 1994; Seifert and Axelrood, 1998), does not include species of Nectria or Neonectria. The present study seems to constitute the first report of Neo. discophora var. rubi on a Rubus species in South America.

ACKNOWLEDGMENTS

The authors thank Gary Samuels (USDA, Maryland), Gustavo Fermín (ULA, Mérida) and Bruno Añez (ULA, Mérida) for their review and comments, and CDCHT-ULA for funding Project FO. 492-01-01.

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