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.

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.

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.