L. A. Álvarez

Comparison of Application Methods of Systemic Fungicides to Suppress Branch Cankers in Clementine Trees Caused by Phytophthora citrophthora

Since 2002, considerable losses of Clementine trees (Citrus clementina) have been observed in Spain due to Phytophthora branch canker of citrus caused by Phytophthora citrophthora. Due to the low efficiency of the available cultural and genetic control measures, application of fungicides is required for economic management of the disease. Fosetyl-Al, metalaxyl, and its enantiomer mefenoxam are the only systemic fungicides registered for control of Phytophthora diseases in Spain. However, their efficacy has not been tested against Phytophthora branch canker. Greenhouse and field experiments were conducted for 3 years in Spain to evaluate the ability of these fungicides and application methods to reduce lesion expansion. Nevertheless, with the inoculation technique used, it was not possible to evaluate the protective activity of fungicides, which can play an important role in their performance under field conditions. None of the fungicide treatments inhibited lesion expansion when applications were made on a curative basis. The residual effect was better on young than on mature trees. Paint treatments were generally more effective in reducing lesion expansion that drip chemigation or foliar sprays. However, this application method is laborious and becomes uneconomical in Spain. Trunk and branch sprays as well as long-term programs of foliar sprays or drip chemigation for control of spring and autumn infections are proposed as targets for future research.

Genetic variation and host specificity of Phytophthora citrophthora isolates causing branch cankers in Clementine trees in Spain

Considerable tree losses have been observed during the past few years in Spain due to Phytophthora branch canker of clementines caused by Phytophthora citrophthora. The emergence of this disease led to the speculation that either the pathogen has evolved increasing its aggressiveness or specificity to clementines. A total of 134 isolates of P. citrophthora collected from 2003 to 2005 in 135 citrus orchards in Spain and 22 reference isolates were analyzed genotypically and phenotypically to determine the structure of the population. Genotypic diversity was evaluated by means of Inter-Simple Sequence Repeat (ISSR) markers. Among the phenotypic characteristics examined, sporangial characters, sexual behavior, growth rates and colony morphology of the isolates at different temperatures were studied. The aggressiveness and host-specificity of selected isolates were evaluated by pathogenicity tests on sweet oranges and clementines under field conditions. Phytophthora branch canker of clementines was associated mainly with one genotype (P-1), which included 88% of the isolates obtained from branches. Strains isolated years before the first disease outbreak clustered also with this major genotype, thus it may be considered as a predominant population. Thirteen other minor genotypes were determined, but most contained only one isolate. Although there was wide variation in the morphological and physiological characters, all Phytophthora isolates obtained from branch cankers were sexually sterile and showed a characteristic petalloid colony pattern. As in previous greenhouse studies, pathogenicity tests under field conditions demonstrated that clementines and their hybrids were more susceptible to P. citrophthora than sweet oranges. However, no evidence was found to support the hypothesis that the emergence of the disease was associated with more aggressive or host-specific forms of P. citrophthora.

First Report of Neofusicoccum parvum Associated with Dieback of Mango Trees in Peru

Mango (Mangifera indica L) is one of the most important cash crops of northern Peru. Since 2003, adult mango trees (cvs. Criollo and Kent) located in Piura Province developed symptoms of dieback characterized by the death of twigs and branches in the tree canopy. Additional disease symptoms involved darkened, elongated lesions on the peduncle, causing an early maturation of the fruit, and in advanced symptoms, stem-end rot of fruits. Symptoms were frequent in the spring months (September to November) when the lesions expand rapidly. Diseased tissues from branches and fruits were collected and small pieces of necrotic tissues were surface disinfected and plated onto potato dextrose agar (PDA) with 0.5 g L-1 streptomycin sulfate. Plates were incubated at 25°C in the dark. All affected tissues consistently developed colonies with a white mycelium, moderately dense, and becoming olivaceous gray after 5 to 6 days. Pycnidia were produced on sterile mango twigs placed on the surface of potato carrot agar (PCA) after 10 days. Conidia were hyaline, guttulate, aseptate, measuring (15-) 18.5 (-22.5) × (4-) 5.2 (-7.5) μm. Conidia became olivaceous and developed one or two septa before germination. Isolates were identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, & A.J.L. Phillips (1). DNA sequences of the rDNA internal transcribed spacer region (ITS) and part of the translation elongation factor 1-alpha (EF1-α) genes were used to confirm the identification through BLAST searches in GenBank (ITS: 99% identity to Accession No. EU080928; EF1-α: 98% identity to Accession No. AY343367). Representative sequences of the studied DNA regions were deposited at GenBank (ITS: Accession No. FJ528596; EF1-α: Accession No. FJ528597). Pathogenicity tests were conducted on 18-month-old potted mango plants cv. Kent with two N. parvum strains (A4 and A5). A mycelial plug (3 cm in diameter) taken from the margin of an actively growing colony of each isolate was put in a wound made with a cork borer of the same diameter on the stem of each plant. Inoculation wounds were wrapped with Parafilm. Controls were inoculated with sterile PDA plugs. Ten replicates for each isolate were used with an equal number of control plants. Plants were maintained in a greenhouse with a temperature range of 22 to 28°C. After 4 weeks, mango plants showed necrotic stem lesions originating from the inoculation point affecting also the branches of the inoculated plants. No differences in lesion area between strains were obtained. No lesions developed in the control plants. Reisolations from necrotic tissues were successful and both isolates were morphologically identical to those used for inoculations. N. parvum was isolated from all symptomatic trees in all surveyed areas. This pathogen has already been reported on mango (2) and currently represents a serious problem in the mango-producing areas of Peru. To our knowledge, this is the first report of N. parvum affecting mango in Peru. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 97:99, 2005.

First report of Phoma exigua var. heteromorpha causing oleander dieback in Spain.

During the autumn of 2003, a new disease was detected in oleander (Nerium oleander L.) nurseries in Valencia and Murcia in eastern Spain. Affected leaves showed ovoid or ellipsoid necrotic spots. Necrotic lesions were also observed on stems and lateral shoots that resulted in severe blight and defoliation. In some cases, severe infections caused the death of plants. Isolations from symptomatic leaves and stems onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate (PDAS) consistently yielded dark olivaceous fungal colonies. For sporulation, these isolates were transferred to potato carrot agar (PCA) and incubated at 25°C for 15 days with a 12-h photoperiod. Abundant pycnidia (200 μm in diameter) developed superficially or immersed in the culture medium. Conidia were hyaline, ellipsoid or cylindrical, guttulate, and occasionally, one septate. Conidial dimensions were 6.1 to 9.6 × 2.2 to 3.2 μm (average 6.2 × 2.8 μm). The addition of a drop of concentrated NaOH to the cultures gave a blue-green pigmentation to the agar changing to brown-red. On the basis of cultural characteristics and fungal morphology, the isolates were identified as Phoma exigua Desmaz. This identification was confirmed by sequencing the complete internal transcribed spacer regions 1 and 2, including the 5.8S ribosomal DNA of isolate Pho 6 (GenBank Accession No. AY899262). This sequence was identical to sequences in GenBank from well-characterized strains of P. exigua (1). Pathogenicity tests were conducted on 9-month-old oleander plants (cv. Splendens Gigantium) using three isolates of P. exigua from different locations. Two methods of inoculation were used. Leaves were spray inoculated with an aqueous suspension (1.5 × 105 conidia per ml) of conidia harvested from 15-day-old cultures grown on PCA, or a 5-mm-diameter agar disc, cut from the margin of an 8-day-old culture growing on PCA, was inserted mycelium side down in a stem wound made with a sterile scalpel and sealed with Parafilm. Controls were inoculated with sterile distilled water or sterile PCA discs. There were five replicates for each isolate and inoculation method with an equal number of uninoculated plants. After inoculation, all plants were covered separately with plastic bags for 48 h to maintain high humidity. Plants were maintained in a greenhouse at 20 to 30°C. Within 5 to 15 days after inoculation, symptoms developed that were similar to those observed in the nurseries. The fungus was reisolated from the stems and leaves of all inoculated plants, completing Kochs postulates. On the basis of ITS sequence data and the host from which they were isolated, our isolates were identified as P. exigua var. heteromorpha (Sch. et Sacc.) Noordeloos et Boerema (2,3). This disease has been previously reported to cause severe damage to oleander in France, California, Italy, and the Netherlands. To our knowledge, this is the first report of P. exigua var. heteromorpha on oleander in Spain. References: (1) E. C. A. Abeln et al. Mycol. Res. 106:419, 2002. (2) M. E. Noordeloos and G. H. Boerema. Versl. Meded. Plziektenk. Dienst. Wageningen 166:108, 1988. (3) H. A. van der AA et al. Persoonia 17:435, 2000.

First report of leaf spot and twig blight of Rhododendron spp. caused by Phytophthora hibernalis in Spain.

During the early spring of 2004, an estimated 20% of containerized nursery stocks of Rhododendron spp. in Asturias (northern Spain) were affected by a foliar disease that has reoccurred annually. Leaf spots were dark brown to almost black, generally oval to round, visible from both sides of the leaf, and expanded to affect the entire leaf including the petiole. Affected leaves abscised from the plant. A Phytophthora sp. was consistently isolated from symptomatic leaf tissues on PARBH medium (3) and hyphal tips were transferred onto potato dextrose agar (PDA). Colonies grown on PDA at 20°C were submerged, had a growth rate of 2.2 mm/day, and had lobes of compact mycelium. Sporangia were semipapillate and caducous with a pedicel (20.0-) 37.7 (-52.5) μm long. Sporangia were asymmetrical in shape with the broadest point near the apex: 25.2 to 40.4 μm long × 10.2 to 15.8 μm wide (average 33.1 × 12.6 μm), and length/width ratio was 2.8:1. Chlamydospores were not observed. Isolates were homothallic and oogonia ranged from 26.5 to 27.5 μm in diameter. Antheridia were mostly amphigynous but occasionally paragynous. Oospores were plerotic and 23.1 to 25.5 μm in diameter. These characteristics conformed to those of Phytophthora hibernalis Carne (2). Sequences of the internal transcribed spacer regions on the isolates and comparison with other sequences in GenBank showed that they were identical to P. hibernalis (Accession No. AY827556.1 from Citrus sp.). For pathogenicity tests, four isolates of P. hibernalis were used to inoculate detached leaves of Rhododendron hybrid Brigitte. The underside of five detached leaves was inoculated with a drop of 40 μL of a suspension of 104 zoospores/ml. Controls were inoculated with a 40-μL drop of sterile distilled water. Leaves were incubated in a moist chamber at 20°C in the dark. A quantification of the lesion area was made 8 days after inoculation using the software Assess-APS. All inoculated leaves developed necrotic lesions that ranged from 0.246 to 1.512 cm2. P. hibernalis was reisolated from infected tissue. Symptoms were not detected on the controls. The test was repeated twice and similar results were obtained each time. P. hibernalis has been described previously as causing brown rot on citrus in Spain (4) and was isolated from rhododendron plants in California and Oregon (1). To our knowledge, this is the first record of P. hibernalis causing foliar blight on Rhododendron species in Spain as well as in Europe. References: (1) C. Blomquist et al. Online publication. doi:10.1094/PHP-2005-0728- 01-HN. Plant Health Progress, 2005. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (3) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986. (4) J. J. Tuset. An. Inst. Nac. Investig. Agrar. Ser. Prot. Veg. N.7, 1977.

First report of Phaeoacremonium parasiticum causing petri disease of grapevine in Peru.

Since 2005, symptoms of grapevine decline have been observed on 4- to 8-month-old grapevines (cvs. Red globe and Crimson) grafted onto 1103 P rootstock in Ica and Pisco valleys in southern Peru. Affected plants exhibited weak growth, interveinal chlorosis, necrosis and wilting of leaves, and death. Dark brown-to-black streaking of the xylem was seen when transverse or longitudinal cuts were made in the trunk and shoots. Symptomatic plants were collected and sections (5 cm long) were cut from the zone between the rootstock and the scion, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally, and small pieces of discolored tissues were placed onto potato dextrose agar (PDA) supplemented with oxytetracycline (500 mg liter-1). Plates were incubated at 25°C in the dark for 15 days. A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and malt extract agar (MEA) in the dark at 25°C for 3 weeks until colonies produced spores (3). Colonies were brown on PDA and olive brown on MEA. Conidiophores were branched, 27.5 to 67.5 (42.5) μm long, and often consisting of a single phialide. Conidia were hyaline, oblong ellipsoidal, 2.5 to 4.5 (3.6) μm long, and 1.2 to 1.9 (1.6) μm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium parasiticum (Ajello, Georg & C.J.K Wang) W. Gams, Crous & M.J. Wingf. (teleomorph Togninia parasitica L. Mostert, W. Gams & Crous) (2,3). Identity of isolate Ppa-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region (Phaeoacremonium-specific primers Pm1-Pm2) with the restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the beta-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. FJ151015). The sequence was identical to the sequence of P. parasiticum (GenBank Accession No. AY328379). Pathogenicity tests were conducted using the isolate Ppa-1. Approximately 20 μl of a suspension containing 103 conidia ml-1 was injected into the pith of four nodes on each of 10 dormant, unrooted, 15 cm long cuttings of cv. Red Globe. Four nodes on each of 10 cuttings were used as controls and injected with an equal volume of sterile distilled water. Inoculation points were covered with Parafilm. The cuttings were planted in plastic pots and maintained at 24 ± 3°C in diffuse light, watering as needed. Within 2 months of inoculation, all P. parasicitum-inoculated cuttings exhibited shoots with very poor growth with small leaves and short internodes. In the xylem vessels, black streaks identical to symptoms observed in declining vines in the vineyard were observed. Control plants did not show any of these symptoms. The fungus was reisolated from internal tissues of symptomatic shoots of all inoculated cuttings but not from the control shoots. To our knowledge, this is the first report of P. parasiticum causing young grapevine decline in Peru. References: (1) A. Aroca and R. Raposo. Appl. Environ. Microbiol. 73:2911, 2007. (2) P. W. Crous et al. Mycology 88:786, 2006. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

Lavender Cotton Root Rot: A New Host of Phytophthora tentaculata Found in Spain

Lavender cotton, Santolina chamaecyparissus, is an evergreen shrub growing primarily in dry, calcareous habitats and is grown in rock gardens and mixed borders mainly for its ornamental and aromatic foliage. During 2004, several commercial nurseries in Valencia Province (eastern Spain) reported high mortality of lavender cotton. The foliage of the diseased plants turned brown, wilted, and died. A Phytophthora sp. was isolated consistently from the soil and roots of infected plants using apple baits and the selective medium PARBH (1), respectively. Four pure cultures (PS-31, PS-32, PS-33, and PS-34) were established from hyphal tips and characterized. Colony morphology on potato dextrose agar (PDA) at 24°C was stoloniferous (short stubby branches) with a growth rate of 2.2 mm per day. Sporangia, chlamydospores, and oospores were produced on V8 agar. The sporangia were ovoid to obpyriform, 27.5 to 64.8 (48.3) × 25 to 52.5 (37.5) μm, length/breadth ratio of 1.3:1, and papillate, from which 20% were caducous with a short pedicel (<5 μm). Hyphal swellings and chlamydospores (22 to 38 μm in diameter) were present. Isolates were homothallic, oogonia were globose, mostly terminal 27.5 to 40 (36.2) μm in diameter, 88% of the antheridia were paragynous, monoclinous, or diclinous, and occasionally with two paragynous antheridia per oogonium. Amphigynous antheridia (12%) were also observed. Oospores were aplerotic, 25 to 35 (32.3) μm in diameter, and thin walled. These characteristics and measurements conformed to the description of P. tentaculata described by Kröber and Marwitz (2). Sequencing the internal transcribed spacer region of Santolina isolates PS-32 and PS-34 and comparison of these sequences with other sequences available in GenBank revealed that they were identical to P. tentaculata (AF266775). Pathogenicity tests used 10 4-to-5-month-old potted lavender cotton and two methods. In the first method, inoculum was prepared on a media of 200 g of oats and 120 ml of V8 juice to 1 liter of distilled water. The medium was inoculated with P. tentaculata grown on PDA and incubated in the dark at 20°C for 4 weeks. Inoculum was buried into the compost mixture around the roots at a rate of 3% (w/v). The second method applied a zoospore drench of 50 ml per plant (1 × 104 zoospores per ml) obtained by inducing zoospores in sterile soil extract from cultures of V8 juice agar. The control plants were inoculated with sterile media and sterile distilled water. The following day, the pots were flooded for 2 days, plants were maintained in a glasshouse at 24 ± 5°C, and watered twice a week. All plants inoculated with the first method had wilted foliage and died within 2 months after inoculation, while plants inoculated with zoospores died after 3 months. P. tentaculata was reisolated and the test was repeated twice. The control plants did not show any symptoms of the disease. P. tentaculata was first reported causing root and stalk rot on Chrysanthemum frutescens hybrids, C. leucanthemum, Delphinium ajacis, and Verbena hybrids in Germany (2). It has also been reported on Verbena hybrids in Spain (3). To our knowledge, this is the first report of P. tentaculata causing root rot on lavender cotton. References: (1) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986. (2) H. Kröber and R. Marwitz. Z. Pflanzenkr. Pflanzenschutz 100:250, 1993. (3) E. Moralejo et al. Plant Pathol. 53:806, 2004.

Identification of Pythium tracheiphilum as the causal agent of vascular necrosis of endive (Cichorium endivia) in Spain

Pythium isolates were recovered from endive plants (Cichorium endivia) showing vascular necrosis collected from commercial fields located in Castellón province (eastern Spain). They were identified as Pythium tracheiphilum on the basis of their phenotypical and molecular profile. Pathogenicity tests conducted with two P. tracheiphilum isolates, obtained from endive and lettuce (Lactuca sativa), respectively, in this region, confirmed that both isolates were pathogenic to endive, with no significant differences in virulence between them. This is the first report of vascular necrosis caused by P. tracheiphilum on endive in Spain.

Bleeding Canker on Mesquite in Peru caused by Phytophthora syringae

Mesquite (Prosopis pallida (Wildenow) Kunth) is a drought-tolerant tree widely distributed in the northern Pacific Coast of South America. This species prevents soil erosion, provides shade, conserves prairies, supports bee nutrition, and provides fruits for human and animal consumption. Since the spring of 2004, bark lesions and bleeding cankers were observed on trunks and branches of 70% of declining mesquite trees in some parks at Ica in southern Peru. Badly affected trees were killed by the disease. Isolations were made from the edge of necrotic lesions of the inner bark and roots using PARPH medium (2) and incubated at 22°C for 7 days. A Phytophthora species was consistently isolated from lesions of 10 mesquite trees, and six pure cultures (PS-87-PS-92) were obtained by transferring hyphal tips and characterized. Colonies were stellate on V8 juice agar (VJA; 2 g CaCO3, 200 ml of V8 juice, and 15 g of agar in 800 ml of distilled water), uniform to slightly radiate on corn meal agar (Oxoid Ltd., London, England), and knotty on PDA (Biokar Diagnostics, Beauvais, France). On VJA at 22°C, the average radial growth rate for the six isolates was 1.7 mm per day. Colonies grew slowly at 5 and 25°C with 0.4 and 0.7 mm per day growth rate, respectively. There was no growth at 30°C. Catenulate hyphal swellings formed on VJA and liquid media (1.5% sterile soil extract). Sporangia were persistent, ovoid to obpyriform, semipapillate with narrow exit pores (<5.0 μm in diameter), 32.3 to 39.7 × 21.0 to 27.2 μm, with a length/width ratio of 1.4:1 to 1.6:1. Sporangia were produced by cutting 5-mm disks from the advancing margin of a colony on VJA and adding disks to 10 ml of 1.5% sterile soil extract for 4 to 5 days at 22°C under fluorescent light. Isolates were homothallic with spherical oogonia, 32 to 35 μm in width with paragynous antheridia, and aplerotic oospores, 26 to 31 μm. These characteristics fit the descriptions of Phytophthora syringae (Kleb.) Kleb. (1). Sequences of the internal transcribed spacer regions on the isolates and comparison with other sequences in GenBank showed that they were identical to P. syringae (Accession No. AJ854297 from Citrus limon). In 2005, two methods were used to inoculate mesquite with two isolates. One method used two 20-mm-diameter branches of five 5-year-old mesquite trees where a 5-mm wound was made with a cork borer and a 5-mm block of the agar culture was placed under the bark and sealed with Parafilm. Another method used 10 4-month-old potted plants that received a 30-ml drench of a 104 zoospores/ml suspension per plant. Controls received clean agar blocks and a sterile water drench for 10 control pots. Two weeks after inoculation, black areas and resinosis were observed around inoculated wounds. Inoculated branches produced cankers of 4.7 to 6.8 cm2, 4 weeks after inoculations. Twenty days after inoculation of roots, wilting and root rots of seedlings occurred. No symptoms were found on the control plants. P. syringae was reisolated from the diseased branches and root rots and pure cultures were established. This test was repeated for both methods with similar results. To our knowledge, this is the first report of P. syringae in Peru and the first description of this pathogen on mesquite worldwide. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (2) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986.