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.

Enhanced production of microsclerotia in recalcitrant Verticillium dahliae isolates and its use for inoculation of olive plants.

AIMS: The optimization of a simple protocol for the mass production of viable microsclerotia (MS) of Verticillium spp., even for recalcitrant isolates, to the inoculation of olive cuttings. METHOD AND RESULTS: Four Verticillium spp. isolates were characterized by growth rate and morphology. Then, the production ability and the viability of MS over time were assessed in seven solid culture media and five aqueous media. The best culture medium, according to the quantity and the quality (size) of the MS produced, was the alkaline-modified sodium polipectate (AMSP) aqueous medium. The MS viability was higher in peat moss substrates. Finally, the MS obtained in this work were infective causing 100% incidence of Verticillium wilt (VW) disease in inoculated olive plants. CONCLUSION: This study demonstrates that the modified sodium polipectate medium amended with 0·1% agar is the most suitable for the production of MS of Verticillium dahliae isolates that have lost the ability to produce MS in standard culture media. SIGNIFICANCE AND IMPACT OF THE STUDY: Mass production of MS for artificial infestation of soil is critical to the study of epidemiological and control aspects of the VW. To overcome the failure in the production of MS in recalcitrant isolates, a culture media was optimized and a successful plant inoculation experiment was carried out with artificial MS.

Selection and evaluation of micro-organisms for biocontrol of Verticillium dahliae in olive.

AIMS: To identify potential biological control agents against Verticillium wilt in olive through a mass screening approach. METHOD AND RESULTS: A total of 47 strains and nine mixtures of micro-organisms were evaluated against Verticillium dahliae in a three stage screening: (i) in vitro, by the effect on the mycelial growth and spore germination of the pathogen; (ii) in natural infested soil, by the effect on the reduction of microsclerotia of the pathogen; (iii) in planta, by the effect on the infection of olive plants under controlled conditions. Various fungal and bacterial strains and mixtures inhibited the pathogen and showed consistent biocontrol activity against Verticillium wilt of olive. CONCLUSION: The screening has resulted in promising fungi and bacteria strains with antagonistic activity against Verticillium, such as two non-pathogenic Fusarium oxysporum, one Phoma sp., one Pseudomonas fluorescens and two mixtures of micro-organisms that may possess multiple modes of action. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a practical basis for the potential use of selected strains as biocontrol agents for the protection of olive plants against V. dahliae infection. In addition, our study presented an effective method to evaluate antagonistic micro-organisms of V. dahliae in olive.

Characterization of a Colletotrichum population causing anthracnose disease on Olive in northern Tunisia.

AIMS: To phenotypically, physiologically and molecularly characterize the causal agent of olive anthracnose in the northern Tunisia and to study its genetic variability and pathogenicity. METHODS AND RESULTS: A total of 43 isolates were obtained from symptomatic olives collected from four regions in northern Tunisia. A range of morphological and physiological characteristics was recorded; and a phylogenetic study, based on the sequence analysis of both internal transcribed spacers and TUB2 gene regions, was performed. Of the 43 isolates, 41 were identified as Colletotrichum acutatum s.s, and only two were affiliated to Colletotrichum gloeosporioides s.s. Two more representative Spanish isolates, included for comparison, were identified as Colletotrichum godetiae. Using six inter-simple-sequence-repeat markers, homogeneity between isolates from different locations and within the same species was recorded. In pathogenicity and virulence studies, C. gloeosporioides s.s was found to be less virulent, while the Spanish C. godetiae isolate was significantly more virulent than the Tunisian C. acutatum s.s. CONCLUSIONS: Olive anthracnose in the North of Tunisia is mainly caused by C. acutatum s.s species. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of olive anthracnose in Tunisia, which combines both phenotypic and molecular approaches. Colletotrichum acutatum s.s group was recorded for the first time in the country as the causal agent of olive anthracnose.

Relative Susceptibility of New Olive Cultivars to Spilocaea oleagina, Colletotrichum acutatum, and Pseudocercospora cladosporioides.

The evaluation of the relative susceptibility of new cultivars to the main diseases of a crop is a key point to consider prior to their release to the nursery industry. This study provides a rigorous characterization of the resistance of 15 new olive cultivars and their genitors ('Arbequina,' 'Frantoio,' and 'Picual') to the three main aerial diseases, peacock spot, anthracnose, and cercosporiosis caused by Spilocaea oleagina, Colletotrichum acutatum, and Pseudocercospora cladosporioides, respectively. To do so, developing leaves and detached green-yellowish fruit were inoculated in laboratory tests with S. oleagina and C. acutatum, respectively, using conidial suspensions of both pathogens. Additionally, a previously validated rating scale was used to assess the incidence of leaves with symptoms of S. oleagina or P. cladosporioides and the fruit rot incidence of C. acutatum in the trees for four years under field conditions. As a result, only two of the cultivars were susceptible to peacock spot, most likely because these new cultivars were previously screened for resistance to the disease on previous phases of the breeding program. Conversely, the 15 cultivars were susceptible or moderately susceptible to cercosporiosis. Five of the 15 new cultivars were classified as resistant to anthracnose, with four of them descendants of 'Frantoio' × 'Picual' crosses. In addition, the cultivars resistance to C. acutatum showed a negative linear correlation with the total phenols content of olive oil. This information regarding disease reaction of the new olive cultivars is essential for nursery industry and growers.

First Report of Botryosphaeria dothidea Causing Fruit Rot of Plum in Spain.

Species in the Botryosphaeriaceae are known to produce cankers, dieback, blights, and leaf spots on many hosts, mainly under stress conditions. Several Botryosphaeria spp. may also cause pre- or post-harvest decay of stone fruit, such as peaches (2). In June 2012, fruit of plum (Prunus domestica cv. Golden Japan) showing soft, brown, and slightly sunken necrotic lesions were observed in several orchards affected by hail in Cordoba province (southern Spain). Symptomatic fruit were collected and incubated at 25°C and 100% relative humidity. Isolations were done on potato dextrose agar (PDA). Mycelium and black pycnidia developed on the surface of incubated fruit and on PDA plates. Conidia were hyaline, aseptate, and fusoid. According to morphological criteria, the fungus was identified as Fusicoccum aesculi, the anamorph of Botryosphaeria dothidea (3). The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS4/ITS5 and sequenced. BLAST analysis of the 528-bp fragment showed 100% homology with the sequence of B. dothidea. Pathogenicity tests were performed on immature healthy fruit (2 weeks before harvest) of the same cultivar from the same orchards. Fruit were washed in deionized water with Tween 20 (Polyoxyethylene 20 sorbitan monolaureate 99%, 0.1 ml/liter) and surface sterilized in 10% sodium hypochlorite for 1 min. Twenty-four fruit were inoculated using mycelial-agar discs. Twelve fruit were previously wounded with a sterile 0.5-mm-diameter needle. The same number of fruit, wounded and unwounded, served as a control. All fruit were incubated at 25°C and 100% relative humidity. Seven days after inoculation, 83% of wounded inoculated fruit showed rot symptoms. After 9 days, fruit rot symptoms started to appear on unwounded inoculated fruit. Twenty days after inoculation, 100% of wounded and unwounded fruit showed rot symptoms that led to mummification of the fruit. Pycnidia developed on inoculated fruit and the fungus was reisolated. No symptoms developed on control fruit. These results demonstrate that B. dothidea is pathogenic on plum and that wounds favor infection, although they are not needed. To our knowledge this is the first report of B. dothidea causing fruit rot of plum in Spain. This pathogen is well known in southern Spain causing a serious fruit rot of olive (1) and could have a great impact on plum production in this region, especially when there is damage to the fruits as occurred this year with hail. References: (1) J. Moral et al. Phytopathology 100:1340, 2010. (2) J. M. Ogawa et al. Compendium of Stone Fruit Diseases. APS Press, St. Paul, MN, 1995. (3) B. Slippers et al. Mycologia 96:83, 2004.

Spilocaea oleagina in Olive Groves of Southern Spain: Survival, Inoculum Production, and Dispersal.

Olive scab caused by the mitosporic fungus Spilocaea oleagina is the most important foliar disease of olive. Limited information is available on pathogen survival and disease epidemiology; however, this information is essential for development of new control strategies. Pathogen survival and inoculum production on infected olive leaves and conidial dispersal were evaluated during 4 years in an olive orchard of the susceptible 'Picual' in southern Spain. Infected leaves in the tree canopy were important for pathogen survival and conidia production. The number of conidia per square centimeter of scab lesion and their viability varied greatly throughout the seasons and between years; conidial density in lesions was highest (about 1 to 5 × 105 conidia cm-2) from November to February in favorable years. Conidial density declined sharply in other periods of the year (becoming zero in summer) or in less favorable years. The pathogen did not form new conidia in scab lesions, although some pseudothecia-like structures and chlamydospores were detected on fallen leaves. Under humid conditions, the pathogen could not be detected on fallen leaves after 3 months because the leaves were colonized by saprophytic fungi. The dispersal of conidia as a function of distance from infected leaves in the tree canopy was well described by an exponential model which, together with the lack of conidia in a Burkard spore trap, showed that conidia were mainly rain-splash dispersed. Some trapped conidia were attached to olive leaf trichomes, suggesting that detached trichomes might enhance wind dispersal of conidia.

Field Resistance to Verticillium Wilt in Selected Olive Cultivars Grown in Two Naturally Infested Soils.

The resistance of 11 olive cultivars to Verticillium dahliae was assessed in two experimental field trials. One-year-old rooted olive cuttings from the World Olive Germplasm Bank (IFAPA research center, Córdoba, Spain) were planted in a heavily infested field in Utrera (Sevilla province) and in a moderately infested field in Andújar (Jaén province) of southern Spain. Plants were assessed for Verticillium wilt resistance during 22 months based on disease severity and tree growth. Severe disease symptoms were observed 6 months after planting in both trials. Twenty months after planting in the heavily infested soil, V. dahliae had killed nearly all of the trees of 'Bodoquera', 'Cornicabra', 'Manzanilla de Sevilla', and 'Picual', demonstrating the elevated risk of planting susceptible cultivars in a soil heavily infested with V. dahliae. 'Arbequina', 'Koroneiki', 'Sevillenca', and especially 'Frantoio', 'Empeltre', and 'Changlot Real' showed a high level of disease resistance. However, all of them were affected by the disease. Although the field results confirmed the level of resistance previously obtained for these olive genotypes under controlled conditions, there were some discrepancies. This information will be useful in managing the disease and also in selecting new cultivars for the breeding of Verticillium wilt resistance.

First Report of Botryosphaeria obtusa as Causal Agent of Olive Tree Branch Dieback in Tunisia.

A branch dieback of olive trees (Olea europaea L. cv. Manzanilla de Sevilla) was observed in 2010 in an orchard (50 ha), located in the Testour region of northern Tunisia. More than 50% of trees were severely damaged by the disease. Symptomatic trees presented dead branches and wilted leaves, which remained attached to the shoots, and the affected tissues appeared abnormally dark compared with the inner bark of healthy branches. Numerous pycnidia were observed on the surface of the infected branches. For diagnosis, symptomatic stems were collected and small pieces of discolored tissues were excised from lesion margins, surface sterilized in 0.5% sodium hypochlorite for 1 min, rinsed and dried on sterilized filter paper, then placed on acidified Difco potato dextrose agar plates (APDA; 2.5 ml of 25% lactic acid per liter). Plates were incubated at 25°C for 4 to 5 days, and hyphal tips from developing fungal colonies were transferred to PDA and placed under fluorescent light (12 h/day). A fastgrowing, pycnidia-producing fungus was consistently isolated, with conidia exuding onto the agar surface of 10-day-old cultures. On the basis of colony characteristics, isolates were identified as Botryosphaeria obtusa (3). Conidia were large, dark brown, aseptate, rounded at both ends or truncate at base, and 25 to 26.8 × 10.5 to 12.03 µm. Pathogenicity tests were performed on detached stems of cv. Manzanilla by 7-mm diameter mycelial plugs cut from actively growing cultures of the fungus. Stems (30 cm length) were cleaned, surface sterilized with sodium hypochlorite (0.25% for 2 min), and wounded with a sterilized scalpel. Mycelial disks were placed over wounds and wrapped with Parafilm to prevent desiccation. Control stems were mock inoculated with sterile agar plugs. Inoculated and control stems were placed in polyethylene boxes and incubated at 25°C. After 45 days, inoculated stems developed brown discoloration, and small dark pycnidia appeared on stem surfaces. Controls remained healthy. Koch's postulates were verified by isolating the fungus from symptomatic stems. To confirm the identification, DNA of one isolate was extracted and the fungal primers ITS1 and ITS4 (4) were used to amplify the internal transcribed spacer region of rDNA. Purified amplicons were sequenced and a BLAST search of the GenBank database revealed 99% homology with B. obtusa isolate HO166525.1. The anamorph of the fungus, Diplodia seriata, has been recognized as the cause of fruit rot of olive (1) and branch canker or dieback (2). To our knowledge, this is the first report of a canker disease of olive trees caused by B. obtusa in Tunisia. References: (1) J. Moral et al. Plant Dis. 92:311, 2008. (2) J. Moral et al. Phytopathology 100:1340, 2010. (3) A. Taylor et al. Australas. Plant Pathol. 34:187, 2005. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Fruit Rot of Olives Caused by Botryosphaeria dothidea in Tunisia.

During the summer of 2010, unfamiliar symptoms of fruit rot were frequently observed on different Tunisian olive (Olea europaea) cultivars. These symptoms appeared to be associated with the damage caused by the olive fruit fly (Bactrocera oleae). At first, infected olives showed a brown color and then fruits begin to depress until they become completely mummified and fall immaturely. This problem was more pronounced on table olive cultivars (Ascolana, Meski, and Picholine) in the northern Tunisian regions (Nabeul), with an infection incidence of 65%. Infected Ascolana olives were disinfected with 70% ethanol for 2 min, rinsed in sterile distilled water, and air dried. Several pieces were cut and placed on acidified (2.5 ml of a 25% [vol/vol] solution of lactic acid per liter of medium) potato dextrose agar medium (PDA). All plates were incubated at 25°C for 4 days under continuous fluorescent light. A fast-growing fungus with an abundant, aerial mycelium, which was gradually veering from white to dark gray, was constantly isolated. On the reverse side of the colonies, an olive green coloration spread to the edge and became darker from the center until the underside was completely black. Conidia produced on the PDA plate were hyaline, single or double cell, ellipsoid, with a subobtuse apex and a truncate base, and averaged 22.70 × 5.32 µm. Conidiophores were hyaline, cylindrical, smooth, branched at the base, with an average of 14 to 24 × 2 to 3 µm. Pathogenicity of an isolate was conducted by dipping 20 olives wounded by a sterilized scalpel in a conidial suspension (105 conidia/ml), covering inoculated olives with moisture filter paper, and incubating them in a polyethylene bag under darkness at 25°C. Controls however, were wounded and dipped in sterile distillated water. Seven days after the inoculation, olives showed a brown color covering half of the fruit. Later (15 days after), this browning was accentuated and several black pycnidia were observed. Forty days after inoculation, fruits were completely dried out and the kernel was already appearing. Controls, however, remained totally healthy. Koch's postulates was then verified and showed that pure cultures were obtained after reisolations from inoculated olives, whereas the controls were free of the fungus. BLAST analysis of the internal transcribed spacer region (ITS) of rDNA of one isolate showed 99% identity with the ITS sequence of Botryosphaeria dothidea (GenBank Accession No. FM955381.1). Species of the family of Botryosphaeriaceae are common pathogens causing fruit rot and dieback of many woody plants (3). Drupe rot problem caused by B. dothidea was reported on olives in Greece (4) and southern Italy (2). It was reported that the fungus invades the drupes through the wounds caused by the olive fruit fly and may even be transmitted by it (1), and recently Moral et al. (3) suggested that the olive fruit fly is essential for the initiation of the disease on the fruit. To our knowledge, this is the first report of fruit rot of olives caused by B. dothidea in Tunisia. References: (1) N. González et al. Bol. San. Veg. Plagas 32:709, 2006. (2) C. Lazzizera et al. Plant Pathol. 57:948, 2008. (3) J. Moral et al. Phytopathology 100:1340, 2010. (4) A. J. L Phillips et al. Mycopathology 159:433, 2005.

First Report of Verticillium Wilt of Pistachio Caused by Verticillium dahliae in Spain.

Pistachio (Pistacia vera L.) trees in the Castilla La Mancha and Andalusia regions of central and southern Spain are grown close to olive orchards, which are often severely affected by Verticillium dahliae. During the last decade, wilt and death of one or several branches have been observed on pistachio (cv. Kerman) scions grafted on rootstock (P. terebinthus). Discoloration of vascular tissue was occasionally observed. In five surveyed orchards, incidence of affected trees was less than 2%. Wood chips with the bark removed from symptomatic trees were washed in running tap water, surface disinfested in 0.5% sodium hypochlorite for 1 min, and placed onto potato dextrose agar plates incubated at 25°C in the dark. Isolates were identified as V. dahliae on the basis of the characteristics of microsclerotia, conidiophores, and conidia. V. dahliae isolate V117 from olive was used as reference (1). The fungus was also isolated from soil in pistachio orchards using wet sieving and a modified sodium polypectate agar medium (1). Inoculum density varied from 0 to 4.73 microsclerotia per gram of soil. P. terebinthus seedlings were inoculated with two isolates of V. dahliae from pistachio trees by injecting the stems with 50 µl of a conidial suspension (107 conidia per ml) (2). Wilt symptoms of varying severity developed in 12 and 15 seedlings of the 20 pistachio seedlings inoculated with each of two isolates. No symptoms developed on the control seedlings. The pathogen was recovered from stem tissues of inoculated plants. To our knowledge, this is the first report of Verticillium wilt of pistachio in Spain. This study demonstrates the susceptibility of certain rootstocks to V. dahliae and the importance of using resistant rootstocks, such as UCBI (2), in pistachio plantations established on soils infested by V. dahliae. References: (1) F. J. López-Escudero and M. A. Blanco-López. Plant Dis. 91:1372, 2007. (2) D. P. Morgan et al. Plant Dis. 76:310, 1992.

Assessing the Susceptibility of Olive Cultivars to Anthracnose Caused by Colletotrichum acutatum.

Selected olive (Olea europaea) cultivars were tested in the field and laboratory for their relative susceptibility to anthracnose caused by Colletotrichum acutatum. A rating scale to assess fruit-rot incidence in naturally infected trees was validated by comparing ratings with direct counts of affected fruit. Fruit-rot incidence varied greatly among 20 cultivars and was correlated with the severity of branch dieback symptoms that developed after fruit-rot epidemics. For determining whether artificial inoculation can be used to predict anthracnose susceptibility in the orchard, detached fruit of 12 cultivars were inoculated with C. acutatum and fruit-rot severity was assessed periodically. Progress of disease severity over time fit the logistic function for all cultivars. The best correlation between fruit-rot incidence in the field and disease severity on inoculated fruit was obtained using a disease susceptibility index that integrated the maximum disease progress rate and the estimated time to reach 50% disease severity. Based on field observations and laboratory data on susceptibility to anthracnose, 21 cultivars were classified into three groups: highly susceptible (Cornicabra, Hojiblanca, Lechín de Sevilla, Manzanilla de Sevilla, Morona, Ocal, Picudo, and Verdial de Huévar); moderately susceptible (Arbequina, Arbosana, Morrut, Pajarero, and Villalonga); and resistant (Blanqueta, Empeltre, Frantoio, Koroneiki, Leccino, Morona-D, Picual, and Razzola). The assessment method may be useful to screen olive cultivars for anthracnose resistance.

First Report of Diplodia seriata, the Anamorph of "Botryosphaeria" obtusa, Causing Fruit Rot of Olive in Spain.

In October, 2006, 1,000 olive fruits (Olea europaea) were collected to study latent infections of Colletotrichum spp., causal agent of anthracnose, in an olive orchard in Córdoba Province (southern Spain). The fruits were incubated at 22 to 24°C and 100% relative humidity. Thirteen percent of the fruits showed a black rot that covered part or all of the fruit and pycnidia formed on the fruit surface. The fungus most consistently isolated from affected fruits was identified as Diplodia seriata, the anamorph of "Botryosphaeria" obtusa, by morphological criteria (1). Pathogenicity tests were performed on 50 immature and 50 mature fruits of olive cv. Hojiblanca. The fruits were washed in sterilized water with Tween 20 at 20 µl/liter and surface sterilized in 20% sodium hypochlorite for 2 min. Twenty-five of the immature and 25 mature fruits were wounded with a sterile 0.5-mm-diameter needle, inoculated by immersion in aqueous conidial suspensions (2.2 × 105 conidia ml-1) for 30 min. The same number of immature and mature fruits, wounded and unwounded, were treated only with sterilized Tween 20 water and served as the control. All fruit were incubated at 22 to 24°C and 100% relative humidity. The experiment was repeated twice. Eight days after inoculation, 75% of the mature inoculated fruits showed a general rot with pycnidia developing on the surface of the fruit. After 20 days, the first symptoms of disease were observed on immature olive fruits (2%) and all mature fruits. After 50 days, only 14% of immature inoculated fruits exhibited symptoms of the disease. The fungus caused mummification of mature fruits. D. seriata was reisolated from lesions on all infected fruits. There were no differences in the amount and severity of disease between wounded and unwounded fruits. Control fruits did not display disease symptoms after 50 days. Incidence of olive fruits affected by D. seriata in olive orchards in southern Spain is unknown, although it may be low since it was detected only in 1 of 12 olive orchards sampled for Colletotrichum spp. in 2006. Incidence of fruit rot associated with D. seriata in this orchard was 1.2% of fruits, which also were affected by Colletotrichum. D. seriata has a worldwide distribution and infects numerous fruit trees. In our research, D. seriata is pathogenic on olive fruits but weakly virulent. To our knowledge, this is the first report worldwide of the anamorph of "B." obtusa causing fruit rot on olives. Reference: (1) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007.

Influence of Fruit Maturity, Cultivar Susceptibility, and Inoculation Method on Infection of Olive Fruit by Colletotrichum acutatum.

Detached olive (Olea europaea) fruit inoculated with isolates of Colletotrichum acutatum, causal agent of olive anthracnose, were used to study host-pathogen interactions. Fruit susceptibility increased with increasing fruit maturity. Wounded fruit were more severely affected than nonwounded fruit; however, the wound effect depended on cultivar and inoculation method. Severity of fruit infection increased with inoculum density, although this effect also depended on fruit maturity and cultivar susceptibility. The susceptibility of selected olive cultivars to anthracnose under field conditions correlated well with the response of immature fruit under controlled conditions. As fruit matured, there were fewer differences among cultivars. Based on these results, an inoculation method using immature green fruit and high inoculum densities (105 to 106 conidia/ml) sprayed on the fruit has been proposed to evaluate olive cultivars for anthracnose resistance under controlled conditions.

First Report of Ascochyta Blight Outbreak of Pea Caused by Ascochyta pisi in Spain.

Characteristic Ascochyta blight lesions were observed on leaves and stems of pea (Pisum sativum L.) 'Dove' grown at two sites in the province of Burgos (northern Spain) during May and June of 2005 and 2006. Mean disease severity of affected tissue reached 47% in 2005 and 72% in 2006. Dark brown, circular, necrotic lesions were sometimes covered with pycnidia. Fungal isolations were made from small pieces of infected tissue by surface disinfecting in 1% NaOCl for 1 min and then washing in deionized, sterile water for 2 min. Tissue pieces were placed on potato dextrose agar (PDA) for 7 days at 20 to 24°C under fluorescents lights with a 12-h photoperiod to induce sporulation. Single-spore isolations were made by streaking conidia from PDA cultures on 2% water agar and picking germinated conidia after 18 h. Fungal colonies grown on PDA and conidia from these cultures were similar to that of Ascochyta pisi Lib., and no chlamydospores or pseudothecia were observed, eliminating the possibility that the isolated fungi were A. pinodes or A. pinodella (3), the other fungi associated with the "Ascochyta complex" of pea. Conidial suspensions (5 × 105 conidia/ml) of two single-spore isolates (Spain-47 and Spain-48) were spray inoculated to runoff on 3-week-old plants of bean (Phaseolus vulgaris L. 'Contender'), chickpea (Cicer arietinum L. 'Blanco lechoso'), lentil (Lens culinaris Medik. 'Pardinar'), pea ('Lincoln'), and faba bean (Vicia faba L. 'Alameda') with 10 replicate plants per isolate. Plants were incubated in a growth chamber at 20 to 24°C and 100% relative humidity (RH) for 48 h and then incubated at the same temperature and 50 to 80% RH for 3 weeks. Characteristic Ascochyta blight lesions were apparent 7 days after inoculation on leaves and stems of pea. No disease symptoms were observed on the other inoculated plants. DNA was extracted from both isolates (Spain-47 and Spain-48) and 610 bp of the glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD) was amplified with gpd-1 and gpd-2 primers (2). Amplicons were direct sequenced on both strands and consensus sequences were aligned. Spain-47 and Spain-48 had identical sequences. A BLAST search of the NCBI nucleotide database with the consensus sequence revealed A. pisi G3PD Accession No. DQ383963 (isolate ATCC 201617, Bulgaria) as the closest match in the database with 100% sequence similarity. These results, coupled with the morphological identification and inoculation results, confirm the identity of the fungus as A. pisi. Although infections by A. pinodes or by unidentified Ascochyta spp. are well known in pea crops in Spain (1), to our knowledge, this is the first report of an outbreak of Ascochyta blight of pea caused by A. pisi under field conditions in Spain. References: (1) M. F. Andrés et al. Patógenos de Plantas Descritos en España. MEC, Madrid, 1998. (2) M. L. Berbee et al. Mycologia 91:964, 1999. (3) E. Punithalingam and P. Holliday. No 334 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1972.

High Susceptibility of Olive Cultivar FS-17 to Alternaria alternata in Southern Spain.

Traditional olive orchards in Spain have been planted at a density of 70 to 80 trees per ha with three trunks per tree. During the last decade, the hedgerow orchard, in which planting density is approximately 2,000 trees per ha, was developed. In 2006 and 2007, we noted a severe outbreak of fruit rot in FS-17, a new cultivar from Italy, in an experimental hedgerow planting in Córdoba, southern Spain. The incidence of fruit rot in 'FS-17' was 80% in January of 2006 and 24% in January of 2007. Cvs. Arbosana, IRTA-i18 (a selected clone from 'Arbequina'), and Koroneiki had no symptoms in either year of the study. Disease incidence in 'Arbequina' was <0.1% only in 2006. Affected fruits were soft with gray-white skin and they eventually mummified. Black-green sporodochia were observed on the surface of diseased fruits. A fungus was isolated from diseased fruits on potato dextrose agar (PDA) and incubated at 22 to 26°C with a 12-h photoperiod. After 8 days of growing on PDA, fungal colonies formed conidial chains having a main axis with up to 10 conidia and secondary and tertiary short branches with two to four conidia. Conidia were obpyriform, ovoid, or ellipsoidal, without a beak or with a short beak, had up to four transverse septa, and measured 11.7 to 24.7 (mean 19.6) µm long and 7.7 to 13.0 (mean 9.6) µm wide at the broadest part of the conidium. The length of the beak of conidia was variable, ranging from 0 to 28.6 (mean 5.5) µm. The fungus was identified as Alternaria alternata (1). Pathogenicity tests were performed by spraying 40 mature fruits of 'FS-17' with a spore suspension (1 × 106 spores per ml). The same number of control fruits was treated with water. After 21 days, inoculated fruit developed symptoms that had earlier been observed in the field. A. alternata was reisolated from lesions on all infected fruits. The fungus was not isolated from any of the control fruits. The experiment was performed twice. The new growing system and the high susceptibility of some olive cultivars, such as FS-17, may result in a high incidence of disease caused by a pathogen that is generally characterized as weakly virulent. To our knowledge, this is the first report of A. alternata causing a severe outbreak of fruit rot on olive trees in the field. References: (1) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002.

First Report of Botryosphaeria ribis as a Branch Dieback Pathogen of Olive Trees in Spain.

Botryosphaeria spp. are known to produce cankers and dieback in several woody hosts. Since 2001, a severe disease resulting in the dieback of branches was observed in olive (Olea europaea) orchards in Andalucía, southern Spain, where the only cultivar affected was Gordal de Sevilla. The affected trees showed abundant dead twigs, and wilted leaves remained attached. These symptoms were similar to those caused by Resseliella oleisuga, a bark borer insect common in the region. Closer inspection showed no evidence of insects, but revealed reddish brown bark lesions that girdled the affected branches. When the outer bark was removed, the affected tissue appeared dark brown, in contrast to the yellowish green of healthy inner bark. A Fusicoccum sp. was consistently isolated from the necrotic bark. Conidia produced in black pycnidia were hyaline, aseptate, fusoid, with a truncate base averaging 19.9 × 6.7 µm, becoming pale brown with all cells the same color, and developing one or two septa when germinated. These characteristics conform to Fusicoccum ribis (1), the anamorph of B. ribis. Pathogenicity of two isolates from symptomatic branches was determined by stem inoculations of 4-year-old cv. Gordal de Sevilla grafted on wild olive trees growing in plastic pots containing a sand/lime/peat soil mixture. Both isolates were inoculated by three techniques. In the first, 7-mm-diameter agar plugs bearing actively growing mycelium were applied to 7-mm-diameter bark wounds made with a cork borer on the middle point of stems (averaging 10 mm in diameter). In the second technique, the same type of inoculum was applied directly to the bark without any wounding. For the third technique, 100 µl of conidial suspension in water (2 × 105 conidia per ml) was applied to bark wounds. The inoculated stem sections were wrapped in Parafilm to retain moisture. All inoculated and control plants (only sterile agar medium or sterile water added) were kept in a greenhouse and watered as needed. There were three replicate plants per isolate and inoculation technique. After 3 months, all plants inoculated with mycelium applied to wounds showed girdling and apical death with browning and wilting of leaves above the inoculation point. In the treatments where mycelium was applied to unwounded bark or where conidial suspensions were applied to wounds, small bark cankers developed to 56 mm long, but neither girdling nor foliar wilting were observed. No symptoms developed in the control plants. There were no significant differences in pathogenicity between the two F. ribis isolates. F. ribis was recovered from necrotic bark from every inoculated plant. These results demonstrate that B. ribis is pathogenic on cv. Gordal de Sevilla olives, and they indicate that bark wounding favors lesion development but is not needed for infection. Reference: (1) B. Slippers et al. Mycologia 96:83, 2004.

Quercus canariensis, a New Host of Trabutia quercina.

Quercus canariensis Willd. is an oak species endemic to northwestern Africa and the Iberian Peninsula. This species is particularly abundant in the southwestern Andalucía Region of southern Spain. During a disease survey in this area from 1997 to 1998, we observed Q. canariensis trees affected by a tar spot disease. Tar spot lesions were clearly differentiated by a black, crustose, and shiny stromata (10 to 20 mm in diameter) on the upper surface of leaves mainly arranged along leaf veins but also scattered randomly over the leaf surface. On most leaves there was little necrosis, and on most trees the damage was not serious, although some trees located in the most humid areas were severely affected with leaf chlorosis and heavy defoliation. Other Quercus species in the area, such as Q. ilex and Q. suber, did not show signs of tar spot. One fungus species was consistently associated with tar spots. The fungus formed ascomata, 250 to 400 µm in diameter, embedded in the stromata. Asci were clavate or saccate, 45 to 55 × 17 to 22 µm, eight spored, and short stalked. The ascus apex was acute to obtuse. Ascospores were arranged irregularly, 20 to 25 (-30) × 5 to 8.5 µm, fusiform to ellipsoidal fusiform, often curved and flattened on one side, thin walled, hyaline, aseptate, and smooth without a gelatinous sheath or appendage. Based on these characteristics, the fungus was identified as Trabutia quercina (Rudolphi ex Fr.) Sacc. & Roum., a pathogen causing tar spot in several Quercus species in the Northern Hemisphere (1). Samples were deposited in the Plant Pathology Mycotheca at the University of Córdoba, Spain (MIC-888 to MIC-897). To our knowledge, this is the first report of T. quercina on Q. canariensis and the first report of this pathogen in Spain. Reference: (1) P. F. Cannon. Mycopathologia 135:69, 1996.

Phytophthora Root Rot of Aleppo Pine Seedlings in a Forest Nursery in Spain.

Since 1993, more than 120,000 ha of former agricultural lands has been forested in southern Spain. However, there is a lack of information about the etiology of the diseases affecting nursery seedlings in the region. During 1998 and 1999, death of Aleppo pine (Pinus halepensis) seedlings was associated with episodes of excess water. The plants were grown in containers in fertilized peat under shade cloth and remained waterlogged for long periods. A few weeks before their scheduled planting in the field, the plants exhibited needle wilting, extensive root necrosis, and root death. Phytophthora species were suspected as the cause of the mortality, and several species of the genus Phytophthora have been reported to cause damping-off of seedlings of Pinus spp. Only P. citrophthora, however, has been associated with Aleppo pine (1). In a nursery in southern Spain, P. drechsleri mating type A2 was isolated consistently from rotted roots of Pinus halepensis seedlings. Fungal identification was based on temperature-growth relationships and sporangial and gametangial characteristics (1). The optimum growth temperature was 24°C, with a rapid decline in growth rate above 30°C. Sporangia were persistent, terminal, nonpapillate, ovoid, and averaged 41 ± 4 × 29 ± 3 µm. The isolates were self-sterile, and antheridia and oogonia were only observed in pairings with A1 testers of P. drechsleri. Oogonia appeared smooth, spherical, and 33 ± 2 µm in diameter, with amphigynous antheridia that were 14 ± 1 µm long. Pathogenicity tests were conducted on 1-year-old seedlings of Pinus halepensis inoculated with two isolates of P. drechsleri collected from Aleppo pine and Quercus ilex seedlings, respectively, and two isolates of P. cinnamomi from Q. ilex. Inoculated and noninoculated control plants were waterlogged 2 days per week. After 18 weeks, both species of Phytophthora produced extensive root necrosis that resulted in needle wilting. Control plants did not develop needle symptoms and only had a low level of root necrosis. No difference in pathogenicity was observed between the isolates of either species. However, disease severity was less with isolates of P. drechsleri from Q. ilex than with those from Aleppo pine. To our knowledge, this is the first report of P. drechsleri causing root rot of Aleppo pine seedlings in forest tree nurseries in Spain and also demonstrates the potential for P. cinnamomi to cause damping-off on this pine species. Reference: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.

First Report of Cylindrocarpon destructans as a Root Pathogen of Mediterranean Quercus Species in Spain.

Cylindrocarpon spp. have been described as producing root rot in Quercus spp. in forest nurseries, mostly when the host plants were weakened (1). To our knowledge, this is the first description of root rot of Mediterranean Quercus (Q. ilex, Q. suber, and Q. faginea) seedlings caused by C. destructans. In spring 2000, heavy mortality of Quercus seedlings was recorded in a nursery in southeastern Spain. The affected plants were 9 months old and growing in the open air in fertilized peat. Symptoms consisted of browning and wilting of leaves, with extensive necrosis of feeder roots. C. destructans (macroconidia averaging 36 × 6 µm, 1 to 3 septate; microconidia 10 × 3 µm; chlamydospores rough, single, or in chains, averaging 9 µm in diameter) was consistently isolated from the necrotic rootlets. Pathogenicity of one isolate from Q. faginea and of a mixture of isolates from the three affected Quercus spp. was determined. Fungal isolates were grown on potato dextrose agar plates for 1 month to allow chlamydospore production, and the inocula were prepared by mixing the contents of three colonized plates with 125 ml of sterile water. Six replicates of 12-month-old Q. ilex and Q. suber seedlings were inoculated by mixing the inoculum from the three plates with the potting medium prior to planting the seedling. All inoculated and control plants (only water added) were grown in a sand-lime-peat soil mixture under greenhouse conditions and watered as needed. After 5 weeks, all the inoculated plants showed extensive root necrosis accompanied by crown symptoms and rapid plant death. No symptoms were evident in control plants, and they produced new rootlets. No significant difference in pathogenicity was noted between the single isolate compared with the mixture of isolates, and the Quercus species did not significantly differ in susceptibility to root rot. C. destructans was easily recovered from necrotic roots. Reference: (1) D. Brayford. Cylindrocarpon. Pages 103-106 In: Methods for Research on Soilborne Phytopathogenic Fungi. American Phytopathological Society, St. Paul, MN, 1992.