Detection of latent infections caused by Colletotrichum sp. in olive fruit.

AIMS: To set up a practical method to detect latent infections of Colletotrichum sp., the causal agent of olive anthracnose, on olives before the onset of disease symptoms. METHODS AND RESULTS: Freezing, sodium hydroxide (NaOH), ethanol and ethylene treatments were evaluated to detect latent infections on inoculated and naturally infected olive fruit by Colletotrichum sp. as non-hazardous alternatives to paraquat. Treatments were conducted using fruit of cultivars Arbequina and Hojiblanca. The disease incidence and T50 were calculated. Dipping in NaOH 0·05% solution and the paraquat method were the most effective treatments on both inoculated and naturally infected fruit, although the value of T50 was lower for the NaOH method than for the paraquat method in one of the experiments. Subsequently, the dipping time in NaOH 0·05% was evaluated. Longer dipping times in NaOH 0·05% were better than shorter ones in cultivar Arbequina, with 72 h being the most effective in cultivar Hojiblanca. CONCLUSIONS: NaOH solution is a practical method to detect latent infections of Colletotrichum sp. on immature olive fruit. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is relevant because we set up a viable, non-hazardous alternative to paraquat to detect latent infections of Colletotrichum sp. using NaOH. The use of NaOH is a simple and eco-friendly tool that allows the determination of the level of latent infections by Colletotrichum in olives. Therefore, our method will be useful in decision-making processes for disease management before the appearance of the first visible symptoms.

Detection of Grapevine Fungal Trunk Pathogens on Pruning Shears and Evaluation of Their Potential for Spread of Infection.

Four vineyards visibly affected by trunk diseases were surveyed at pruning time in 2012 and 2013 in Spain, to determine whether pruning tools are capable of spreading grapevine trunk diseases from vine to vine. In each vineyard, pruning shears were regularly rinsed with sterile water, collecting liquid samples for analysis. Molecular detection of grapevine fungal trunk pathogens (GFTPs) was performed by nested polymerase chain reaction using specific primers to detect Botryosphaeriaceae spp. Eutypa lata, Cadophora luteo-olivacea, Phaeoacremonium spp., and Phaeomoniella chlamydospora. All of these GFTPs, with the exception of E. lata, were detected in samples from the four vineyards, C. luteo-olivacea and Phaeoacremonium spp. being the most prevalent. Co-occurrence of two, three, or four different GFTPs from the same sample were found, the simultaneous detection of C. luteo-olivacea and Phaeoacremonium spp. being the most prevalent. In addition, fungal isolation from liquid samples in semiselective culture medium for C. luteo-olivacea, Phaeoacremonium spp., and P. chlamydospora was also performed but only C. luteo-olivacea was recovered from samples collected in three of four vineyards evaluated. Pruning shears artificially infested with suspensions of conidia or mycelial fragments of C. luteo-olivacea, Diplodia seriata, E. lata, Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora were used to prune 1-year-old grapevine cuttings of '110 Richter' rootstock. Successful fungal reisolation from the cuttings 4 months after pruning confirmed that infested pruning shears were able to infect them through pruning wounds. These results improve knowledge about the epidemiology of GFTPs and demonstrate the potential of inoculum present on pruning shears to infect grapevines.

First Report of Phaeoacremonium venezuelense Associated with Wood Decay of Apricot Trees in Spain.

In July 2011, a survey was conducted to evaluate the phytosanitary status of apricot trees (Prunus armeniaca L.) in an orchard in Binissalem (Mallorca Island, Spain). Fungal isolation was performed on a 40-year-old apricot trees (cv. Galta Vermella, double-grafted onto bitter almond and Japanese plum) showing a collapse of branches, chlorosis of leaves, and shoot dieback. These symptoms appeared in approximately 10% of the trees. Black spots and dark streaking of the xylem vessels were observed in cross- or longitudinal sections of the branches. Symptomatic branches were collected and wood sections (10 cm long) were cut, washed under running tap water, surface-disinfested 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 plated onto malt extract agar (MEA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Dishes were incubated at 25°C in the dark for 14 to 21 days, and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues (more than 50% of the isolations). Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were brownish orange on PDA and MEA. Conidiophores were short and occasionally branched, and 26 to 35 (avg. 29) µm long. Phialides were terminal or lateral, mostly monophialidic. Conidia were hyaline, oblong-ellipsoidal or fusiform-ellipsoidal, 3 to 4 (avg. 3.9) µm long, and 1 to 1.5 (avg. 1.2) µm wide. Based on these characters, the isolates were identified as Phaeoacremonium venezuelense L. Mostert, Summerb. & Crous (2,3). DNA sequencing of a fragment of the beta-tubulin gene of the isolate 9.3 using primers T1 and Bt2b (GenBank Accession No. KF765487) matched P. venezuelense GenBank accession HQ605026. Pathogenicity tests were conducted using isolate 9.3. Ten 2-year-old apricot trees of cv. Galta Rotja grown in pots were wounded in two branches with a 8-mm cork borer. A 5-mm mycelium PDA plug from a 2-week-old culture was placed in the wound before being wrapped with Parafilm. Ten control plants were inoculated with 5-mm non-colonized PDA plugs. Plants were maintained in a greenhouse at 25 to 30°C. Within 5 months, shoots on all Phaeoacremonium-inoculated branches had weak growth with chlorosis of leaves and there were black streaks in the xylem vessels. The vascular necroses that developed on the inoculated plants were 5.5 ± 0.6 cm long, significantly greater than those on the control plants (P < 0.01). Control plants did not show any symptoms. The fungus was re-isolated from discolored tissue of all inoculated cuttings, completing Koch's postulates. P. venezuelense was reported as a pathogen of grapevines in Algeria (1) and South Africa (2) and, to our knowledge, this is the first report of P. venezuelense associated with wood decay of apricot trees in Spain or any country in the world. References: (1) A. Berraf-Tebbal et al. Phytopathol. Mediterr. 50:S86, 2011. (2) L. Mostert et al. J. Clin. Microbiol. 43:1752, 2005. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

Fungal trunk pathogens associated with wood decay of almond trees on Mallorca (Spain).

Severe decline of almond trees has recently been observed in several orchards on the island of Mallorca (Balearic Islands, western Mediterranean Sea). However, the identity of the causal agents has not yet been investigated. Between August 2008 and June 2010, wood samples from branches of almond trees showing internal necroses and brown to black vascular streaking were collected in the Llevant region on the island of Mallorca. Several fungal species were subsequently isolated from the margin between healthy and symptomatic tissue. Five species of Botryosphaeriaceae (namely Botryosphaeria dothidea, Diplodia olivarum, D. seriata, Neofusicoccum australe and N. parvum), Eutypa lata, Phaeoacremonium iranianum and Phomopsis amygdali were identified based on morphology, culture characteristics and DNA sequence comparisons. Neofusicoccum parvum was the dominant species, followed by E. lata, D. olivarum and N. australe. First reports from almond include D. olivarum and Pm. iranianum. Two species are newly described, namely Collophora hispanica sp. nov. and Phaeoacremonium amygdalinum sp. nov.

First Report of Cylindrocladiella parva and C. peruviana Associated with Black-foot Disease of Grapevine in Spain.

From 2007 to 2009, Cylindrocladiella-like isolates were recovered from grapevine (Vitis vinifera L.) roots with symptoms of black-foot disease in Spain, where the causal agents of this disease have been previously reported as Campylocarpon and Cylindrocarpon species (1,2). Three representative isolates were selected to confirm their identity: CPa1 and CPa2 from Asturias (northern Spain), and CPe523 from Cuenca (central Spain). Isolates were incubated on malt extract agar (MEA) and Spezieller Nährstoffarmer Agar (SNA) with carnation leaves (4) at 25°C for 10 days in darkness. On MEA, colonies developed light brown, cottony mycelium. On SNA, all three isolates produced chlamydospores in chains, and conidia were zero-to one-septate, but CPa1 and CPa2 produced longer conidia (10.4 to 18.9 [15.3] × 1.7 to 3.1 [2.4] µm) than CPe523 (6.4 to 12.3 [9.7] × 1.6 to 3.3 [2.4] µm). A fragment of the beta-tubulin gene from all isolates was sequenced with primers T1 and Bt2b (1) and deposited in GenBank (Accession Nos. JQ693133, JQ693134, and JQ693135). CPa1 and CPa2 showed high similarity (99%) to Cylindrocladiella parva (AY793486) and CPe523 showed high similarity (99%) to C. peruviana (AY793500), which is in agreement with the corresponding morphological features of these species (4). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seed soaked for 12 h in 300 ml of distilled water and autoclaved three times. Inoculum was prepared by inoculating two fungal disks (8 mm in diameter) of a 2-week-old culture of each isolate grown on potato dextrose agar to wheat seed and incubation at 25°C for 4 weeks. One-month-old grapevine seedlings were planted individually in 220-cc pots filled with a potting medium of sterilized peat moss and 10 g of inoculum, and grown in the greenhouse at 25°C in a completely randomized design. Controls were inoculated with sterile, noninoculated wheat seed. There were six replicate plants per isolate, with an equal number of controls, and the experiment was repeated once. Symptoms developed in all plants by 20 days post-inoculation and consisted of reduced vigor, necrotic root lesions, and occasionally mortality, all of which resembled the symptoms from grapevines in the field from which the isolates were originally recovered. Mean shoot dry weights of inoculated plants (0.25, 0.16, and 0.28 g for CPa1, Cpa2, and CPa523, respectively) were significantly lower (P < 0.05) than that of the controls (0.74 g). Mean root dry weights of inoculated plants (0.28, 0.16, and 0.29 g for CPa1, Cpa2, and CPa523, respectively) were also significantly lower (P < 0.05) than that of the controls (0.68 g). Isolates recovered from the roots of inoculated plants were identical morphologically and molecularly to C. parva and C. peruviana, thereby satisfying Koch's postulates. No symptoms were observed on the control plants. These Cylindrocladiella spp. have been reported from nurseries or vineyards in South Africa and New Zealand (3). To our knowledge, this is the first report of C. parva and C. peruviana associated with black-foot disease of grapevine in Spain, and in Europe. References: (1) S. Alaniz et al. Plant Dis. 91:1187, 2007. (2) S. Alaniz et al. Plant Dis. 95:1028, 2011. (3) E. E. Jones et al. Plant Dis. 96:144, 2012. (4) L. Lombard et al. Mycol. Progress DOI 10.1007/s11557-011-0799-1, 2012.

First Report of Campylocarpon fasciculare Causing Black Foot Disease of Grapevine in Spain.

In May 2008, symptoms of black foot disease were observed on 8-year-old grapevines (Vitis vinifera L.) cv. Garnacha in Albuñol (Granada Province, southern Spain). Affected plants showed delayed budding with low vigor. Roots showed black discoloration and necrosis of wood tissues. Root fragments were cut, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discolored or necrotic tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Plates were incubated at 25°C in the dark for 10 days and all colonies were transferred to PDA. A Cylindrocarpon-like fungus was consistently isolated from necrotic root tissues. Single conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer Agar (SNA) and incubated at 25°C for 10 days in darkness. On PDA, the isolates developed white, thick, and cottony to felty abundant mycelium. On SNA, all isolates produced slightly to moderately curved one-septate (22.5-) 25.6 (-27.5) × (5-) 5.63 (-6.25) µm, two-septate (30-) 36.1 (-45) × (6.25-) 7.08 (-7.5) µm, three-septate (37.5-) 47.9 (-52.5) × (6.25-) 7.5 (-8.75) µm, four-septate (47.5-) 53.3 (-62.5) × (7.5-) 7.89 (-8.75) µm, and five-septate (52.5-) 61.8 (-67.5) × (7.5-) 8 (-8.75) µm macroconidia. Microconidia were not observed. DNA sequence of the rDNA internal transcribed spacer region (ITS) was obtained for isolate Cf-270 and deposited in GenBank (Accession No. HQ441249). This sequence showed high similarity (99%) to the sequence of Campylocarpon fasciculare Schroers, Halleen & Crous (GenBank Accession No. AY677303), in agreement with morphological features (1). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seeds that were soaked for 12 h in flasks filled with distilled water. Each flask contained 300 ml of seeds that were subsequently autoclaved three times after excess water was drained. Two fungal disks of a 2-week-old culture of C. fasciculare (isolate Cf-270) grown on PDA were placed aseptically in each flask. The flasks were incubated at 25°C for 4 weeks and shaken once a week to avoid clustering of inoculum. Plastic pots (220 cm3) were filled with a mixture of sterilized peat moss and 10 g of inoculum per pot. One-month-old grapevine seedlings were planted individually in each pot and placed in a greenhouse at 25 to 30°C in a completely randomized design. Control plants were inoculated with sterile uninoculated seeds. Six replicates (each one in individual pots) were used, with an equal number of control plants. The experiment was repeated. Symptoms developed on all plants 20 days after inoculation and consisted in reduced vigor, interveinal chlorosis and necrosis of the leaves, necrotic root lesions with a reduction in root biomass, and plant death. The fungus was reisolated from the roots of affected seedlings and identified as C. fasciculare, completing Koch's postulates. No symptoms were observed on the control plants. Black foot disease of grapevines can be caused by different species of Cylindrocarpon and Campylocarpon. C. fasciculare was first reported in South Africa in 2004 (1). To our knowledge, this is the first report of C. fasciculare causing black foot disease of grapevine in Spain as well as other countries in Europe. Reference: (1) F. Halleen et al. Stud. Mycol. 50:431, 2004.

First Report of Cylindrocarpon liriodendri on Kiwifruit in Turkey.

During a routine survey of diseases of kiwifruit (Actinidia chinensis Planch.) cv. Hayward conducted in autumn of 2009 in Ardesen, Rize Province (eastern Black Sea Region, Turkey), symptoms of a new disease were observed in five locations. Affected trees showed leaf wilting that frequently led to the death of the trees. Symptoms at ground level included necrotic lesions on woody tissues of both the rootstock and roots. Small pieces from necrotic wood and root tissues were surface disinfested and plated onto potato dextrose agar (PDA) medium amended with 0.5 g liter-1 of streptomycin sulfate and incubated for 7 days at 25°C in the dark. Isolates were transferred to PDA and presumptively identified as a Cylindrocarpon sp. by morphology and conidial characteristics. The isolates were transferred to PDA and Spezieller Nährstoffarmer Agar (SNA) and then incubated at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed floccose to felted mycelium, which varied in color from brown-yellow to sepia. On SNA, all isolates produced microconidia measuring 6.25 to 15 (9.6) × 2.5 to 5 (3.02) µm and macroconidia of one-septate measuring 7.5 to 20 (13.3) × 2.5 to 5 (3.8) µm, two-septate measuring 12.5 to 25 (20.7) × 3.25 to 5 (4.58) µm, and three-septate measuring 16.3 to 30 (11.04) × 3.75 to 5 (4.82) µm. Chlamydospores 7.5 to 11.3 (9.78) µm were intercalary or terminal in the mycelium, single or occasionally in chains. Identity of these isolates was determined by a multiplex PCR system using a set of three pairs of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a product size of 253 bp, which is characteristic of Cylindrocarpon liriodendri J.D. MacDonald and E.E. Butler, in agreement with morphological features (2). Additionally, the internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolates 10K-TR1 and 10K-TR2 and deposited in GenBank (Accession Nos. HQ113122 and HQ113123). These sequences showed high similarity (98%) with the sequence of C. liriodendri (GenBank Accession No. DQ718166). A pathogenicity test was conducted using isolate 10K-TR1 and repeated twice. Six 8-month-old callused and rooted cuttings of kiwifruit cv. Hayward were surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, washed twice with sterile distilled water (SDW), and inoculated by dipping their roots for 30 min in a spore suspension of the fungus (1 × 106 conidia ml-1) obtained from 30-day-old colonies grown on PDA. Six control cuttings were dipped in SDW. Two weeks later, cuttings were drench inoculated with 50 ml of the designated spore suspension to guarantee root infection and controls were drenched again with SDW. Plants were maintained in a greenhouse with a temperature range of 25 to 30°C. Four months after inoculation, the inoculated plants developed wilting and root symptoms similar to those observed in natural infections and C. liriodendri was reisolated, completing successfully Koch's postulates. No symptoms were observed on the control plants. To our knowledge, this is the first report of C. liriodendri on kiwifruit trees in Turkey. References: (1) S. Alaniz et al. Plant Dis. 93:821, 2009. (2) F. Halleen et al. Stud. Mycol. 55:227, 2006.

First Report of Damping-Off Caused by Cylindrocarpon pauciseptatum on Pinus radiata in Spain.

In the fall of 2009, damping-off of Pinus radiata seedlings was observed in a pine nursery in Sant Feliu de Buixalleu, Girona Province, northeastern Spain. Plants exhibited needle blight, extensive root necrosis, and root death. Root sections of symptomatic plants were cut, washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small fragments of discolored tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Plates were placed at 25°C in the dark for 10 to 14 days, and all fungal colonies were transferred to PDA. A Cylindrocarpon sp. was consistently isolated from necrotic root tissues. Single-conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer agar (SNA) (2) at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed abundant mycelium, which varied from white-to-grayish brown or golden brown. On SNA, all isolates produced two-septate, (35-) 39.4 (-40) × (7.5-) 7.7 (-8.75) µm, and three-septate, (32.5-) 40.9 (-52.5) × (7.5-) 7.7 (-8.75) µm, macroconidia. Microconidia, one-septate macroconidia, and chlamydospores were not observed. Identity of these isolates was determined by a multiplex PCR system using a set of three pair of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a 117-bp product that was characteristic of Cylindrocarpon pauciseptatum Schroers & Crous. Morphological characteristics also supported this identification (4). Internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolate 1052 and deposited in GenBank (Accession No. HQ441248). This sequence was identical (100%) with the sequence of C. pauciseptatum (GenBank Accession No. HM036590). Pathogenicity tests were conducted with inoculum produced on wheat kernels that were soaked in distilled water in flasks for 12 h. Each flask contained 200 ml of kernels that were subsequently autoclaved three times after excess water was drained. Two fungal disks from a 2-week-old culture of C. pauciseptatum (isolate 1052) grown on PDA were placed aseptically in each flask. Cultures in flasks were incubated at 25°C for 4 weeks and shaken once a week. A plastic pot (220 cm3) was filled with a mixture of sterilized peat moss and 10 g of inoculum. A 1-month-old seedling of P. radiata was planted in plastic pots and placed in a greenhouse at 25 to 30°C in a completely randomized design with six replications. Controls contained sterile wheat kernels. The experiment was repeated. Symptoms developed 20 days after inoculation and consisted of root lesions, a reduction in root biomass, needle blight, and the death of all seedlings. The fungus was reisolated from affected seedlings. Damping-off was not observed on the control plants. C. pauciseptatum causing black foot disease of grapevine (3) was first found in Spain in 2008, but to our knowledge, this is the first report of C. pauciseptatum causing damping-off of P. radiata in Spain. References: (1) S. Alaniz et al. Plant Dis. 93:821, 2009. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, 2006. (3) M. T. Martin et al. Plant Dis. 95:361, 2011. (4) H. J. Schroers et al. Mycol. Res. 112:82, 2008.

Evaluation of Vineyard Weeds as Potential Hosts of Black-Foot and Petri Disease Pathogens.

Weeds were sampled in grapevine rootstock mother fields, open-root field nurseries, and commercial vineyards of Albacete, Alicante, Castellón, Murcia, and Valencia provinces in Spain between June 2009 and June 2010 and evaluated as potential hosts of black-foot and Petri disease pathogens. Isolations were conducted in the root system and internal xylem tissues for black-foot and Petri disease pathogens, respectively. Cylindrocarpon macrodidymum was successfully isolated from the roots of 15 of 19 weed families evaluated and 26 of 52 weed species. Regarding Petri disease pathogens, one isolate of Phaeomoniella chlamydospora was obtained from Convolvulus arvensis, and three isolates of Cadophora luteo-olivacea were obtained from Bidens subalternans, Plantago coronopus, and Sonchus oleraceus. Pathogenicity tests showed that Cylindrocarpon macrodidymum isolates obtained from weeds were able to induce typical black-foot disease symptoms. When inoculated in grapevines, isolates of Cadophora luteo-olivacea and Phaeomoniella chlamydospora were also shown to be pathogenic on grapevine cuttings. Our ability to recover grapevine pathogens from vineyard weeds and to demonstrate pathogenicity of recovered strains on grape suggests that these weeds may serve as a source of inoculum for infection of grapevine.