Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

Cedeño, Luis; Carrero, Chrystian; Quintero, Kleyra; Pino, Henry; Espinoza, Wilmer destructans var. destructans and neonectria discophora var. rubi associated with black foot rot on blackberry (rubus glaucus benth.) in Merida, Venezuela Interciencia, vol. 29, núm. 8, agosto, 2004, pp. 455-460 Asociación Interciencia Caracas, Venezuela

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative 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

SUMMARY

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

Introduction H. Schrenk, (anthracnose; Ce- als in order to avoid or dimin- unaffected mature and imma- deño and Palacios, 1991), ish the incidence and/or dis- ture fruits. Some mummified In Venezuela blackberry (Ru- Sphaerotheca macularis (Wallr.: semination of the pathogens, fruits were greenish brown, bus glaucus Benth.) production Fr.) Lind, (powdery mildew; the future of blackberry pro- while others were dark brown takes place in the Aragua, Cedeño et al., 1995a), Pero- duction in these regions is in to almost black. The roots and Barinas, Lara, Mérida, Táchira nospora sparsa Berk. (downy doubt. the crown of the dead plants and Trujillo States. Although in mildew; Cedeño et al., 1995b), In May 1999, a disease of were affected by a black rot the context of the national agri- Coniothyrium fuckelii Sacc. unknown cause was discovered (Figure 1). Transverse cracks cultural system this minor fruit- (cane blight; Cedeño and in a commercial field of were seen in the roots, and in bearing species does not have Carrero, 2000) and Botrytis ci- blackberry located in the vil- them the cortex came off with an outstanding economic and nerea Pers.: Fr. (gray mold), lage of Monterrey at El Valle, ease, revealing light brown, social importance, in the which reduce the yields, alter Municipality Libertador, dark brown and black streaks Andean region, particularly in the quality of the fruit and di- Mérida State. Plants were af- in the vascular tissues. In the the Mérida State, it is popular minish the productive life of fected by a die-back disease. internal portion of the crown and represents an important the plants. Because of the high The dying plants showed of dying plants, black streaks primary source of revenue for incidence of these pathogens, symptoms of wilt, and the were also seen, which con- small and medium size produc- the current amount of black- dead ones had lost all their trasted with the yellowish ers. Their fruits are consumed berry in the regional market leaves. In some leaves of the white color of the surrounding fresh and processed in diverse does not satisfy the demand; dying plants necrosis was vis- healthy tissues. All the dead ways as syrup, ice creams, moreover, the fruit is of very ible only at the tip and the plants had a hollow central marmalades, juice, wines and low quality and tends to spoil borders, while others were portion at the crown. Later, yogurt. quickly. completely dry and brittle; all similar symptoms were de- The main natural problems Without concerted efforts on the leaves were strongly curled tected in plants grown in sec- of this crop in the Venezuelan the part of the Autonomous toward the upper surface. tors of Macho Capaz (Munici- Andes are the diseases caused Agricultural Health Service The dying floricanes had pality Campo Elias) and La by the fungi Glomerella cingu- (SASA) and growers to remove mummified fruits (dry and Azulita (Municipality Andrés lata (Stoneman) Spaulding. & and destroy all infected materi- hard), in addition to apparently Bello).

KEYWORDS / Black foot rot / Blackberry / Cylindrocarpon / Neonectria / Received: 04/21/2004. Modified: 07/02/2004. Accepted: 07/16/2004.

Luis Cedeño. Agronomist, Univer- (ULA), Mérida, Venezuela. Ad- ment, ULA. Researcher, Labo- Wilmer Espinoza. Agricultural sidad de Oriente, Venezuela. dress: IIAP-ULA. P.O. Box. 77 ratory of Phytopathology, Technician, General Agronomy Master in Plant Pathology, Uni- (La Hechicera), Mérida 5101- IIAP-ULA. Section, IIAP-ULA. versity of Georgia, USA. Re- A, Venezuela. Kleyra Quintero. Phytotechnician, searcher, Laboratory of Phyto- e-mail: [email protected] Assistant, Laboratory of Phyto- pathology, Instituto de Investi- Chrystian Carrero. Agronomist, pathology, IIAP-ULA. gaciones Agropecuarias (IIAP), Universidad del Zulia, Venezu- Henry Pino. Phytotechnician, Fruit Universidad de Los Andes ela. Master in Forest Manage- Trees Section, IIAP-ULA.

AUG 2004, VOL. 29 Nº 8 0378-1844/04/08/455-06 $ 3.00/0 455 RESUMEN

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

RESUMO

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

In February and May 2002, Materials and Methods Difco). Additionally, perithecia formed in situ and in vitro. In on roots and crowns of plants that had been briefly surface both cases, the cultures were dead by black foot rot at Isolation and identification of disinfested with 0.5 NaOCl, incubated at room temperature Miraflores and Tabay, respec- the fungal pathogens were placed directly on AWA. (22ºC) and 12h of light from a tively, clusters of reddish to Following the procedure of lamp placed at 45cm (2 tubes reddish brown perithecia were Isolations were made from Hansen and Smith (1932), 10 of fluorescent daylight F40D commonly found (Figure 2). symptomatic roots and crowns monoconidial and 10 monoas- Extralife, Former 40W, and 2 The anatomical and morpho- collected in sectors of El cosporic cultures were pro- tubes of black light Roblan logical features of perithecia Arado, La Azulita, Macho duced, respectively, using one 110V, BLB 40W). were characteristic of mem- Capaz, Miraflores, Monterrey of the numerous mass isolates The identification was car- bers of the Order Hypocre- and Tabay, all located in obtained from infected roots ried out comparing the infor- ales, Subdivision Ascomyco- Mérida State. After washing the and crowns collected in the mation registered with those tina (Gerlach and Nilssen, material with running tap water sectors where perithecia were published in the specialized lit- 1963; Rossman, 1983; Sa- for 1h, small pieces (ca. 2- not found, and from ascospores erature (Booth, 1966, 1967; muels and Brayford, 1990; 3mm) were taken from the in- exuded by perithecia developed Samuels and Brayford, 1990; Rossman et al., 1999; Bray- terface of healthy and diseased on PDA. The parameters evalu- Brayford et al., 2004). Fifty ford et al., 2004). In Tabay, it tissues; these immediately were ated in the monoconidial cul- microconidia, macroconidia or was common to observe a surface sterilized with 0.5% so- ture were the morphology of chlamydospores were measured violet pigmentation on the dium hypochlorite (NaOCl) for the colonies on PDA and the in each monoconidial culture, basal portion of dying canes 3min, rinsed three times in form and size of the asexual while in each monoascosporic (Figure 3), while the roots sterile distilled water (SDW), reproductive structures (micro- isolate 50 macroconidia, 25 and the crown of the dead dried on sterile absorbent paper conidia, macroconidia and perithecia, and 50 ascospores plants showed black rot and then plated onto Petri chlamydospores) produced in were measured. symptoms. dishes containing water agar vitro, while in the monoascos- The present work was acidified (pH 4.5) with lactic poric cultures they were the Growth rate carried out with the purpose acid (AWA). The dishes were morphology of the colonies on of establishing the identity incubated at 25 ±1ºC in the PDA, the form and size of the The selected cultures were of the microorganisms caus- dark and, later, the emergent asexual structures (macro- grown in plates containing ing the described symptoms, colonies were transferred to test conidia) developed in vitro, and 20ml of potato-sucrose agar and to evaluate their patho- tubes containing slants of po- those of the sexual structures (PSA; Booth, 1966), cornmeal genicity. tato-dextrose agar (PDA; (perithecia and ascospores) agar (CMA; Difco) or PDA,

456 AUG 2004, VOL. 29 Nº 8 -2 which were inoculated with radial growth (mm/day) of the Pathogenicity tests 15lb·in during 3 consecutive disks (6mm diam) taken from mycelium was calculated mea- days. The roots of 3½ months 10-days-old colonies on 1.2% suring in each colony 2 per- For the inoculation tests with old seedlings were placed on water agar medium. Five plates pendicular diameters, substract- the monoconidial culture, the substratum and the roots of each substratum were used ing the diameter of the initial blackberry seedlings 6-8cm tall carefully separated, while the and they were incubated for 7d inoculum and dividing the re- were used. They were grown in foliage came out through a cir- at 22ºC in the darkness. The sult by two. black polyethylene bags con- cular hole that included the taining soil sterilized for 1h cover and the base of the dish. with dry heat at 110ºC during Ten seedlings were inoculated 3 successive days. Before in- by wounding and ten with no oculation the seedlines were wounds, applying to each one placed in a human chamber 5ml of a monoconidial suspen- with the purpose of preventing sion (1.62 macroconidia/ml) on stress resulting from transplant- the root system. As a control 3 ing. The inoculum was pro- wounded seedlings and 3 un- duced by growing the fungus wounded seedlings were used in test tubes (25x150mm) con- and SDW was applied on taining barley grains, prepared them. The dishes were sealed as follows. A layer (2-3cm) of with a double layer of moistened sterile cotton was Parafilm® perforated with a dis- placed in the bottom of each section needle to allow aera- tube and barley grains were tion, covered with aluminum placed on top of the cotton to foil to prevent illumination of a height of 2cm. The tubes the roots, and finally placed in were sealed with cotton pro- vertical position during 3d un- Figure 1. Symptoms (in nature) of black foot rot caused by Cy- tected with gauze and sterilized der the lamp (12:12h light/dark lindrocarpon destructans var. destructans. twice, with an interval of 24h, regime). Later, the seedlings for 20min at 121ºC and were transferred to the green- 15lb·in-2. Each tube was inocu- house, where they were wa- lated with four disks (6mm tered twice per week with a diam) of mycelium taken from nutrient solution (potassium ni- a 5-days-old culture grown on trate 0.5g; Epsom salts 0.5g; PDA. Cultures were incubated di-Ammonium phosphate at room temperature (22ºC) and 0.16g; calcium nitrate 0.89g; under 12:12h (light:dark) re- water up to 1000ml). gime for 30d. The inoculum All the inoculation tests were was applied at 1.5% (w/w) and carried out twice. Isolations each seedling received a 1:50 were made from the seedlings dilution in SDW. Seedlings infected experimentally to used as control only received a prove the Koch postulates. suspension of barley grains not colonized by the fungus. The Results inoculum was covered with soil and the seedlings were trans- Isolation and identification ferred to the greenhouse where of fungal pathogens Figure 2. Perithecia of Neonectria discophora var. rubi (in situ). they remained covered with bags of transparent plastic for Only two fungi were isolated 3d. Starting 3d after inoculation from infected crowns and roots (dai), the seedlings were exam- of R. glaucus, and from perith- ined periodically to evaluate the ecia. Based on the morphology development of foliage symp- of the asexual reproductive toms. Regularly, some seed- structures produced in situ and lings were examined to observe in vitro, they were recognized damages on the roots and on as different species of Cylin- the neck. drocarpon Wollenw. (Booth, The pathogenicity tests of 1966). the monoascosporic cultures One of the fungi isolated were done in 9cm disposable consistently formed micro- Petri dishes (Unestam and conidia, macroconidia, and Stenström, 1989) containing a chlamydospores. It was ob- substratum made of granulated tained from all the materials vermiculite, dolomitic lime and coming from the different geo- peat (VT-M Premier Sogemix, graphical locations sampled, Vegetable Transplant Growing except from Miraflores and Figure 3. Violet stain on the neck of canes naturally infected by Mix, pH 7.0), which was steril- Tabay. Based on the type, mor- Neonectria discophora var. rubi. ized for 1h at 121ºC and phology and size of asexual

AUG 2004, VOL. 29 Nº 8 457 structures produced in vitro, the side the substratum forming in creamy white colored var. ianthothele Wollenw., and fungus was identified as C. de- terminal and intercalary chains masses deposited around the Neo. discophora (Booth, structans var. destructans and they were spherical to el- ostiole. The anatomical and 1966, Mantiri et al., 2001, (Zinssm.) Scholten [=C. radici- liptic in shape, hyaline at the morphological characteristics, Brayford et al., 2004). cola Wr.] (Booth, 1966; Seifert beginning and then with thick as well as the size of the per- and Axelrood, 1998), whose cellular golden brown walls, ithecia and ascospores that it Pathogenicity tests teleomorph, Neonectria radici- 12.9 (11.3-14.5µm) x 11.4 contained, coincided with cola (Gerlach & Nilsson; (10.1-12.7µm) in diameter, those described for Neonec- The inoculation tests with Mantiri & Samuels [= smooth, although deposits of tria discophora (Mont.) C. destructans var. destructans radicicola Gerlach & Nilsson] substances that made them ap- Mantiri & Samuels [=Nectria resulted in the production on (Gerlach and Nilsson, 1963; pear rough were frequently ob- mammoidea W. Phillips & seedlings with the same symp- Booth, 1967; Samuels and served on the surface. Plowr. var. rubi (Osterw.) toms as observed in the field. Brayford, 1990; Mantiri et al., The teleomorph of C. de- Weese] (Brayford et al., When the plastic bags were 2001) was discovered in Swe- structans was not found in situ 2004), teleomorph of C. ian- removed, all the seedlings den on rotten leaves, peduncles and was not produced in vitro, thothele var. ianthothele growing in contaminated soil and bulbs of Cyclamen per- which suggests that it is a het- Wollenw., which differs from had leaflets showing wilt sicum L. (Gerlach and Nilsson, erothallic species. C. destructans in not produc- symptoms and tip necrosis. 1963). The second Cylindrocarpon ing microconidia or chlamy- The leaflets had lost their On PSA, HMA and PDA fungus was isolated from roots, dospores (Booth, 1966). The natural green color and media, the mycelial growth of crowns and perithecia taken identity of the species was showed the tendency to curl C. destructans var. destructans, from diseased plants sampled confirmed by Gary J. Samuels toward the upper surface. The averaged 3.2, 2.6 and 2.3 mm/ in sectors of Miraflores and (Personal Communication). necrosis began at the tip and day, respectively. On PDA the Tabay. The cultures originating On PSA, HMA and PDA, at the borders, at first light colonies produced cottony from perithecia always formed the mycelial growth of the se- brown and later becoming aerial mycelium that was gray- perithecia on PDA. Clusters of lected monoascosporic cul- dark brown. Lightly sunken ish-white at first, later becom- perithecia were found on roots tures averaged 1.3, 0.5 and light brown, dark brown, vio- ing cream-colored to light with black rot (Miraflores) and 0.6mm/day, respectively. On let, purple to black lesions brown. From the reverse, the on the neck of canes with die- PDA the colonies showed a were observed on the roots. cultures were reddish-brown in back (Tabay). Perithecia were distinctive violet pigmentation The leaflets of the youngest the central portion and beige in ovoid to spherical, reddish to similar to that observed on leaves were the first to die. the borders. When the dishes reddish-brown, 380-700 x 350- the neck of the canes infected Some roots were completely were opened a distinctive odor 650µm, smooth and shining, naturally in Tabay. The colo- blackened, collapsed and had of musty earth was detected. with a slightly protruding, nies were floccose and the lost the cortex. Of the seed- The microconidiophores de- domed and darkened apical surface mycelium was violet lings, 60% died at 7dai and velop as lateral phialides or ter- ostiole through which as- while the aerial mycelium the rest died during the fol- minally on short lateral cospore were extruded in white was yellowish brown; the lowing two weeks. Seedlings branches which also may form or buff tendrils. The perithecial colony reverse was dark. inoculated with the control one or more laterals, each ter- wall did not show a pseudo- The monoascosporic cul- were unaffected. C. destruc- minating in one or more cylin- parenchymatous structure but tures also developed abundant tans var. destructans was the drical to awl-shaped phialides. was composed of an intertwin- macroconidia on violet-col- only microorganism consis- The phialides measured 18.0- ing network of thickened hy- ored sporodochia, but no mi- tently isolated from the seed- 35.0 x 2.5-3.0µm. The micro- phae. Within the perithecia croconidia or chlamydospores, lings experimentally infected, conidia were hyaline, cylindri- paraphyses and hyaline, cylin- and strands of golden to dark which demonstrates that it is cal to oval and 7.2 (5.6-8.8µm) drical asci containing 8 as- brown hyphae. The conidiog- the cause of black rot on the x 3.9 (3.3-4.5µm). The macro- cospores in uniseriate disposi- enous cells were cylindrical, roots and crown of blackberry, conidiophores are produced as tion, were also observed. The hyaline, with a collarette and R. glaucus. lateral branches with elongated ascospores were hyaline, ellip- thickened ring in the apex, The pathogenicity tests with stipe and loosely branched soid to subfusoid with a simple and arose at the tips of short, Neo. discophora var. rubi were apex, each branch terminating septum, 11.6 (11.0-13.0µm) x much branched conidiophores. also successful. Five days after in one or more phialides. The 5.0µm, flat when young, but The macroconidia were hya- inoculation, in 70% and 40% macroconidia were hyaline, cy- faintly spinulose and pale line, curved, cylindrical with of the seedlings inoculated lindrical or slightly wider at the brown at maturity. round ends, 0-5 septate. Al- with and without wounding, distal end, straight or curved After 6 weeks of growth on most 90% of the conidia were respectively, the younger with rounded ends and a protu- PDA, all the monoascosporic 3-5 septate, being the 4-sep- leaves had died and other berant basal scar, 1-3 septate, cultures had produced fertile tate the most abundant. The leaves showed tip necrosis and although occasionally some perithecia similar to those 3-, 4- and 5- septate macro- tended to curl towards the up- with 4 or 5 septa were ob- found in the field. The perith- conidia averaged 52.6x6.8, per surface. The foliage of the served. The 1-septate macro- ecia developed mainly in 64.8x7.1 and 71.9x7.3µm, control seedlings was not af- conidia measured 26.1 (23.1- clusters that, in general, respectively. fected. One week later, 70% 29.2µm) x 5.8 (5.2-6.2µm) and shared the same stromatic The absence of micro- and 60% of the seedlings with the 2-3 septate measured 37.4 base and they showed the conidia and chlamydospores, and without wounding, respec- (34.5-40.3µm) x 7.4 (6.8- same transition in color from together with the morphology tively, had severe foliage dam- 8.0µm). The 5-septate macro- white, pale yellow, bright red, and size of the macroconidia age, while the control seed- conidia averaged 37.0 (36.0- dark red, amber to finally al- and violet color of the colo- lings remained unaffected. 38.0µm) x 7.0µm. The chlamy- most black as seen in nature. nies confirm the identity of Five and one-half weeks after dospores appeared on and in- The ascospores were extruded this species as C. ianthothele inoculation, 50% of the

458 AUG 2004, VOL. 29 Nº 8 wounded seedlings and 50% groups, each distinguished by gen of stressed plants and kills the root tissues of of those without wounds were the presence or absence of mi- (Brayford, 1991). plants affected by stress caused dead, while the control seed- croconidia and/or chlamy- C. destructans is a cosmo- by transplant, root prune, lings remained healthy. The dospores. The species included politan natural inhabitant of anaerobiosis around the roots, roots of the dying seedlings in the first group produce the soil, commonly associated shade produced by high planta- showed the same violet color abundant microconidia but do with roots and residues of a tion density and inappropriate as was observed on the neck not have mycelial chlamy- wide variety of woody and use of certain pesticides of canes affected by die-back dospores. In the second group herbaceous plants (Booth, (Kluge, 1966; Evans et al., in Tabay. Around the neck were placed those species that 1966, 1967; Kluge, 1966; 1967; Petäistö, 1982; Unestam only few roots turned violet. do not form microconidia nor Samuels and Brayford, 1990), and Stenström, 1989; Unestam The affected roots presented chlamydospores. Those of the especially in alkaline soils et al., 1989; Beyer-Ericson et the following sequence of col- third group develop micro- and, less frequently, in soils al., 1991). The stress caused by ors: violet, light brown, dark conidia and chlamydospores. of coniferous forests (Matturi O2 deficiency due to an excess brown and black. The roots of Those of the fourth group and Stentöm, 1964). This fun- of water in the soil (Unestam dead seedlings had collapsed have chlamydospores but do gus is considered to be a et al., 1989), or by the continu- and blackened, and C. ian- not produce microconidia. necrotrophic and opportunistic ous use of fungicides (Unestam thothele var. ianthothele was Booth (1966) concluded that pathogen, because it only ex- et al., 1989; Beyer-Ericson et frequently isolated from the the name C. radicicola was presses its pathogenicity on al., 1991), weaken the root sys- roots artificially infected. antedated by C. destructans plants subjected to stress or tem facilitating the infection by and located it in group 3; when conditions favor its de- C. destructans. Apparently, the Discussion while he placed in group 2 the velopment (Unestam et al., soil reaction is not a critical anamorphs of Neo. discophora 1989; Brayford, 1991). How- factor because the fungus The fungi species that for- var. discophora (=N. mammoi- ever, in this study, the se- grows substantially at pH be- merly were considered in the dea) and Neo. discophora var. lected monoconidial strain of tween 2.9 and 7.9 (Unestam et genus Nectria are now located rubi (=N. mammoidea var. the fungus infected and rotted al., 1989). The toxin seems to in the families and rubi). The var. rubi was re- the roots of healthy, un- have antibiotic effects on other Bionectriaceae (Rossman et tained as a variety of Neo. dis- stressed seedlings, indicating fungi (Kluge, 1966; Unestam et al., 1999). Nectria and Neo- cophora because it has only that it is a true pathogen of al., 1989) because it inhibits nectria are included in the been found on plants of the R. glaucus. the growth of saprophytes on Nectriaceae (Rossman et al., genus Rubus (Brayford et al., C. destructans is very sensi- infected roots and, also, it 1999, Mantiri et al., 2001). 2004); however, the tendency tive to antagonism and to seems it has antibiotic activity Species of Nectria s. str. in- in var. rubi is to produce as- competition on the roots and in vitro against Trichoderma clude secondary pathogens of cospores in culture and macro- because of that, in order for it viride and other fungi (Kluge, trees; they have red, anatomi- conidia that are somewhat to be able to compete success- 1966). cally distinctive perithecia and shorter and wider than those fully, it has to invade and be- C. destructans possesses anamorphs in the genus Tuber- of the var. discophora, and to come dominant in the weak- strains that differ widely in cularia. The species of Neo- grow slower in vitro. Whether ened roots before the arrival of pathogenicity (Kluge, 1966). nectria are easily distinguished var. discophora and var. rubi saprophytes (Unestam et al., There is an intimate correla- from those of Nectria because can be separated at the species 1989). In normal and biologi- tion between the production of of their Cylindrocarpon level will require a more in- cally balanced soil, C. destruc- toxin and the degree of viru- anamorphs; their perithecia are tense comparative study using tans does not become patho- lence (Kluge, 1966; Unestam anatomically and morphologi- DNA sequences. genic (Kluge, 1966), but when et al., 1989), and between cally different. The only species of Cylin- the microbial community of virulence and the production On the basis of analyses of drocarpon that has been asso- the soil is altered by treatment in vitro of a dark-colored pig- mitochondrial ribosomal DNA ciated with damage (canker with water steam or fungi- ment (Unestam et al., 1989). sequences (Mantiri et al., and die-back) on Rubus plants cides, toxins produced by the The correlation between toxin 2001; Brayford et al., 2004), grown in Europe (Brayford, fungus might accumulate at production and pathogenicity the species of Nectria with 1991) is C. ianthothele var. toxic levels that favor the in- indicates that the substance, Cylindrocarpon anamorphs ianthothele whose teleomorph, fection of the host plants. The probably of phenolic nature, were placed in the genus Neo- Neo. discophora var. rubi, was severity of the attack by C. plays an important role in the nectria, including Neonectria discovered for the first time in destructans on Pinus sylvestris development of the root rot discophora and Neonectria Switzerland on rotten roots of L. is increased when the con- that affects the seedlings of P. radicicola. Previously, Nectria raspberry, R. ideaus, (Oster- tinuous use of fungicides in- sylvestris (Kluge, 1966). Ac- radicicola had been included walder, 1911). Later, this mi- hibits the action of the antago- cording to Ahn and Lee in the -group croorganism was found in as- nists (Unestam et al., 1989). (2001), the virulence of the C. (Samuels and Brayford, 1990) sociation with similar damage C. destructans is a “pioneer destructans that attacks gin- and Nectria was included in on raspberry in Scotland colonizer” of roots because it seng (Panax quinquefolius L.), the Nectria mammoidea-group (Alcock, 1925) and England has the capacity to grow and is controlled by a double helix (Booth, 1959, 1966; Rossman, (Nattrass, 1927; Pethybridge, develop quickly in atmo- viral RNA.

1983; Brayford et al., 2004). 1927). However, pathogenicity spheres with low O2 levels that The pathogenicity tests evi- Each of these groups was of the fungus has not been are not favorable for other denced that both of the iso- characterized by perithecial demonstrated experimentally fungi (Ludeking and Relab lated fungi are pathogens of R. anatomy and by anamorphs. (Alcock, 1925; Nattrass, 1927; den Haan, 2002). glaucus, but Neo. radicicola Booth (1966) recognized 27 Pethybridge, 1927) and be- The pathogenicity of C. de- was more aggressive, as it species of Cylindrocarpon and cause of that it has been con- structans is related to the pro- killed more seedlings in a distributed them among four sidered as a secondary patho- duction of a toxin that weakens shorter time. This difference in

AUG 2004, VOL. 29 Nº 8 459 pathogenicity could be related with fungicides. The slow de- vices. Gainesville, Florida, USA. Kluge E (1966) Pathogenität ge- to the rate of growth, since on velopment of the infection in- 1114 pp. genüber kiefernsämlingen und Cylindrocar- Beyer-Ericson L, Damm E, Unestam toxinbildung bei PSA, PDA and CMA, Neo. duced by this fungus could ex- pon radicicola Phytopathol. T (1991) An overview of die- Wr. radicicola grew 2.5, 3.8 and plain why Neo. discophora has Z. 55 back and its causes in Swedish : 368-388. 5.2 times faster than Neo. dis- always been found associated forest nurseries. Eur. J. For. Ludeking D, Relab den Haan BV cophora. with old blackberry plants, Path. 21: 439-443. (2002) Cylindrocarpon destruc- The growth rate of Neo. dis- while Neo. radicicola has been Booth C (1959) Studies of tans. www.denhaan.nl/02maren. cophora was very low in all isolated from seedlings and Pyrenomycetes. IV. Nectria (Part htm the substrata used, as compared from young and adult plants. I). Mycol. Papers 73: 1-115 Mantiri F, Samuels GJ, Rahe JE, to that of Neo. radicicola. The This is apparently the first Booth C (1966) The Genus Cylindro- Honda B (2001) Phylogenetic disease caused by Neo. disco- report of C. destructans var. carpon. Commonwealth Mycol- relationship in Neonectria spe- phora, thus, progresses more destructans as a cause of black ogy Institute. Kew, Surrey, En- cies having Cylindrocarpon gland. 56 pp. anamorphs inferred from mito- slowly than that induced by rot on the roots and the crown chondrial ribosomal DNA se- Neo. radicicola and, conse- in a species of Rubus. How- Booth C (1967) Nectria radicicola. quences. Can. J. Bot. 79: 334- quently, it requires that the ever, it is important to point C.M.I. Descriptions of Patho- 340. genic Fungi and Bacteria Nº148. conditions that facilitate and fa- out that perithecia of Neo. 2 pp. Matturi ST, Stenstöm H (1964) Dis- vor their occurrence converge radicicola var. radicicola occur tribution and status in the soil and remain in place for longer in native and disturbed vegeta- Brayford D (1991) Nectria canker of of Cylindrocarpon species. raspberry. In Compendium of Trans. Brit. Mycol. Soc. 47: periods. In this respect it is im- tion in Venezuela. Samuels and Raspberry and Blackberry Dis- 577-587. portant to point out that the Brayford (1990) reported speci- eases and Insects. APS Press. symptoms and signs of the dis- mens collected in 1971 in the St. Paul, Minnesota, USA. p. Nattrass RM (1927) Notes on 20. Nectria rubi. II. Trans. Br. ease caused by Neo. disco- mountains of Nirgua (Carabobo Mycol. Soc. 12: 23-27. Brayford D, Honda BR, Mantiri FR, phora, in general, have only State, Dumont-VE 1525, on Osterwalder A (1911) Über eine been observed during later unknown vine), and in La Samuels GJ (2004) Neonectria and Cylindrocarpon: the Nectria neue auf kranken Himbeer- lapses to very humid years Carbonera (Mérida State; mammoidea group and species wurzeln vorkommende Nectria (Nattrass, 1927; Pethybridge, Dumont-VE 2512, non identi- lacking microconidia. Mycologia und die geörige Fusarium gen- 1927) or very dry years fied wood). These specimens 96: 572-597. eration. Ber. Dtsch. Bot. Ges. 29: 611-622. (Brayford, 1991). In 1988 a se- are preserved in the crypto- Cedeño L, Carrero C (2000) Conio- vere attack of Neo. discophora gamic herbarium of the New thyrium fuckelii causando quema Petäistö RL (1982) Risk of fungal occurred on the red raspberry York Botanical Garden. The lit- en cañas de mora de Castilla. infection on coniferous seed- Fitopatol. Venez. 13: 38-40. lings after root pruning in for- of Scotland (Brayford, 1991), erature related to the fungi that est nurseries. Folia For. 505: 1- after a previous year whose se- attack Rubus grown in America Cedeño L, Palacios-Prü EL (1991) 8. Antracnosis en mora de Castilla vere summer compacted soil (Farr et al., 1989; Alfieri et al., (Rubus glaucus Benth) causada Pethybridge GH (1927) Notes on around the primocanes that 1994; Seifert and Axelrood, por Glomerella cingulata en Nectria rubi. I. Trans. Br. damaged the tissues at the 1998), does not include species Venezuela. Fitopatol. Venez. 4: Mycol. Soc. 12: 20-23. point of attachment to the of Nectria or Neonectria. The 17-20. Rossman AY (1983) The Phrag- crown. In Tabay, Mérida, Ven- present study seems to consti- Cedeño L, Carrero C, Quintero K mosporous Species of Nectria ezuela, a region where the fun- tute the first report of Neo. dis- (1995a) Primer reporte en Ven- and Related Genera. Mycologi- gus appeared associated with cophora var. rubi on a Rubus ezuela de Sphaeroteca macularis cal Papers Nº150. Common- causando mildiú polvoriento en wealth Agriculture Bureaux. 11% of dead plants, the soil species in South America. mora de Castilla (Rubus glau- Kew, Surrey, England. 164 pp. remained very humid for a cus). Rev. Forest. Venez. 1: 20. Rossman AY, Samules GJ, Rogerson long time; while in Miraflores ACKNOWLEDGMENTS Cedeño L, Carrero C, Quintero, K CT, Lowen R (1999) Genera of the disease was observed after (1995b) El mildiú lanoso, Pero- Bionectriaceae, Hypocraceae three months without rain. In The authors thank Gary nospora sparsa, de la mora de and Nectriaceae (, December 2003, the disease Samuels (USDA, Maryland), Castilla (Rubus glaucus) en Ven- Ascomycetes). Stud. Mycol. 42: 1-248. was seen in adult plants that Gustavo Fermín (ULA, Mérida) ezuela. Rev. Forest. Venez. 1: 45. died in the Institute of Agricul- and Bruno Añez (ULA, Méri- Evans G, Cartwright JB, White NH Samuels GJ, Brayford D (1990) tural Research of Universidad da) for their review and com- (1967) Production of a phyto- Variation in Nectria radicicola toxin, necrolide, by some root and its anamorph Cylindrocar- de Los Andes, located in Santa ments, and CDCHT-ULA for surface isolates of Cylindrocar- pon destructans. Mycol. Res. Rosa, Mérida, after several funding Project FO. 492-01-01. pon radicicola Wr. Plant and 94: 433-442. months of abundant precipita- Soil 26: 253-260. Seifert KA; Axelrood, PE (1998) tion. It is possible that the REFERENCES Farr DF, Bills GF, Chamuris GP, Cylindrocarpon destructans var. pathogenicity of this microor- Rossman AY (1989) Fungi on destructans. Fungi Canadensis ganism is related to an excess Ahn IP, Lee YH (2001) A viral plants and plant products in the Nº337. Can. J. Plant Pathol. double-stranded RNA up regu- United States. APS Press. St. 20: 115-117. of humidity after damages Paul, Minnesota, USA. 1252 pp. caused by the wind followed lates the fungal virulence of Unestam T, Stenström E (1989) A Nectria radicicola. Mol. Plant- Hansen HN, Smith RE (1932) The method for observing and ma- by water logging (Brayford, Microbes Interact. 14: 496-507. mechanism of variation in im- nipulating roots and root asso- 1991) or with stress caused by Alcock NL (1925) A note on rasp- perfect fungi: Botrytis cinerea. ciated fungi on plants growing long lapses of deficiency or berry canker (Nectria rubi Phytopathol. 22: 953-964. in nonsterile substrate. Scand. J. For. Res. 4: 51-58. abundance of water in the soil. Osterwalder). Trans. Proc. Bot. Gerlach W, Nilsson L (1963) It is our opinion that in the Soc. Edinburgh 29: 197-198. Beiträge zur kenntnis der gat- Unestam T, Beyer-Ericsson L, sites under study, the infection Alfieri SA, Langdon KR, Kimbrough tung Cylindrocarpon Wr. V. Strand M (1989) Involvement takes place through the wounds JW, El-Gholl NE, Wehlburg C Nectria radicicola n. sp. die of Cylindrocarpon destructans bisher unbekannte hauptfrucht- in root death of Scotch pine caused by pruning since they (1994) Diseases and Disorders of Plants in Florida. Bulletin form von Cylindrocarpon radici- seedlings: pathogenic behavior are not commonly treated for Nº14. Florida Department of cola Wr. Phytopathol. Z. 48: and predisposing factors. Scand. healing nor are they protected Agriculture and Consumers Ser- 251-257. J. For. Res. 4: 521-535.

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