Thaer Yaseen

Real-time detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg μl-1 for P. nicotianaeand 100 pg μl−1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg μl-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.

Seasonal Fluctuations of Sap-Feeding Insect Species Infected by Xylella fastidiosa in Apulian Olive Groves of Southern Italy

Abstract A study on seasonal abundance of Auchenorrhyncha species and their infectivity by Xylella fastidiosa in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were monitored monthly over a 2-yr period from five olive groves. A total of 15 species were captured, identified, and tested for presence of X. fastidiosa by polymerase chain reaction (PCR). For three species, Philaenus spumarius L., Neophilaenus campestris (Fallèn), and Euscelis lineolatus Brullé, positive reactions to X. fastidiosa were obtained, on average, in 16.3, 15.9 and 18.4% of adult insects, respectively. Philaneous spumarius was the dominant species (39.8% of total Auchenorrhyncha captured) with the highest adult abundance in summer months. Adult P. spumarius and N. campestris were first detected between March and May in both years, and all insects tested during these periods (year 1: n = 42, year 2: n = 132) gave negative reactions to X. fastidiosa by PCR. Similarly, first adults of E. lineolatus that appeared from October to November (year 1: n = 20, year 2: n = 15) tested negative for presence of X. fastidiosa. Given the lack of transstadial and transovarial transmission of X. fastidiosa and considering that P. spumarius is univoltine, control measures against nymphal stages of P. spumarius should be investigated as means of population suppression to reduce spread of X. fastidiosa in olive groves.

First Report of Citrus Root Rot Caused by Phytophthora palmivora in Egypt

During spring-summer 2009, a survey was conducted to determine the species of Phytophthora present in citrus nurseries in Egypt. A total of 300 samples of soil and fibrous roots were collected from the rhizosphere of symptomatic Volkameriana lemon (Citrus volkameriana Tan. & Pasq.) plants growing in Delta (Benha-Qalyubia) and a desert (Cairo/Alexandria desert road) citrus nurseries. Plants showed various symptoms. Canopies of affected plants showed few and yellowish leaves, a general stunted growth, no new vegetation, and sometimes sudden desiccation; the root system showed few dark brown feeder roots, no new yellow-white apexes, and a fibrous appearance of the rootlets due to disintegration of the cortical bark but not of the xylem. Collected rootlets and soil were plated in Petri dishes containing a selective medium for the oomycete Phytophthora (2) and incubated for 3 to 6 days at 19 ± 1°C as described by Ippolito et al. (1). Pure cultures were obtained by single-hypha transfers and the isolates were identified as Phytophthora palmivora (Butler) Butler on the basis of morphological and cultural characteristics (3). Isolates formed stoloniferous colonies on potato dextrose agar (PDA) and grew between 10 and 30°C, with the optimum at 25°C. On V8 juice agar, they showed a highly fluffy pattern and produced terminal and intercalary globose chlamydospores. Sporangia were papillate, elliptical (45 to 51 × 29 to 35 μm; length/breadth ratio of 1.3:1.8), and were caducous with short pedicel. All isolates were A2 mating type, forming spherical oogonia and amphigynous antheridia in dual cultures with reference P. palmivora isolate of A1 mating type. Identification of the isolates was further confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region using the universal primers ITS4 and ITS6. BLASTn analysis of ITS sequences (GenBank Accession No. HE583183) showed 99% homology with P. palmivora isolates available in GenBank. Pathogenicity tests for P. palmivora were conducted by inoculating three groups of ten 6-month-old Volkameriana lemon plants, transplanted into 1.4 liter pots with growing medium artificially inoculated at the rate of 1% (v/v) of P. palmivora inoculum produced according to Yaseen (4). Ten uninoculated plants served as a control. Two months after the inoculation, plants were analyzed for canopy symptoms and the presence of pathogen in feeder roots. More than 50% of inoculated plants showed foliage symptoms and extensive decay of feeder roots. Colonies of Phytophthora were recovered from necrotic rootlets and identified as P. palmivora, fulfilling Kochs postulates. To the best of our knowledge, this is the first report of P. palmivora as a pathogen to citrus plants in the Egyptian nurseries. P. palmivora should be considered a potential threat to the Egyptian citrus industry since it may negatively influence the nurseries and orchards production in the future. References: (1) A. Ippolito, V. De Cicco, and M. Salerno. Rivista di Patologia Vegetale 2:57, 1992. (2) H. Masago, M. Yoshikawa, M. Fukada, and N. Nakanishi. Phytopathology 67:425, 1977. (3) D. J. Stamps. Revised tabular key to the species of Phytophthora. CAB International Mycological Institute, Kew, Surrey, 1990. (4) T. Yaseen. Molecular diagnosis and biological control of Phytophthora-citrus root rot. PhD thesis. University of Bari, Italy, 2004.

Development of Real-Time Isothermal Amplification Assays for On-Site Detection of Phytophthora infestans in Potato Leaves

Real-time loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed targeting the internal transcribed spacer 2 region of the ribosomal DNA of Phytophthora infestans, the potato late blight causal agent. A rapid crude plant extract (CPE) preparation method from infected potato leaves was developed for on-site testing. The assays specificity was tested using several species of Phytophthora and other potato fungal and oomycete pathogens. Both LAMP and RPA assays showed specificity to P. infestans but also to the closely related species P. andina, P. mirabilis, P. phaseoli, and P. ipomoeae, although the latter are not reported as potato pathogen species. No cross-reaction occurred with P. capsici or with the potato pathogens tested, including P. nicotianae and P. erythroseptica. The sensitivity was determined using P. infestans pure genomic DNA added into healthy CPE samples. Both LAMP and RPA assays detected DNA at 50 fg/μl and were insensitive to CPE inhibition. The isothermal assays were tested with artificially inoculated and naturally infected potato plants using a Smart-DART platform. The LAMP assay effectively detected P. infestans in symptomless potato leaves as soon as 24 h postinoculation. A rapid and accurate on-site detection of P. infestans in plant material using the LAMP assay will contribute to improved late blight diagnosis and early detection of infections and facilitate prompt management decisions.

First Report of Verticillium Wilt of Mango (Mangifera indica) Caused by Verticillium dahliae in Italy

Mango (Mangifera indica L.) is an important fruit crop in many tropical and subtropical countries. This crop has been recently introduced in Italy, mainly in Sicily (southern Italy), where it proves to be a good commodity for the national market. However, the future of mango cultivation in Sicily is threatened by diverse biotic and abiotic factors, which may limit production and fruit quality. For this reason, an investigation into soilborne diseases of mango was carried out in summer 2009 and spring 2010 (1). During May 2009, typical symptoms of Verticillium wilt were observed in four young mango orchards, cv. Kensington Pride, in Catania Province. The symptoms observed included gradual leaf yellowing, marginal browning, slow growth, and dieback on one side of the shoots. The dead leaves remained attached to infected branches and no defoliation was observed. Cross sections of affected branches showed brown vascular discoloration. The incidence of infected trees was about 25% of 96 examined trees. Small pieces of brownish, vascular wood tissues were surface disinfested and placed onto potato dextrose agar (PDA). After 5 days of incubation at 25°C in the dark, the isolates were purified using the single-spore isolation method. Pathogen identification was initially based on morphological characteristics, and then confirmed by molecular methods. The colonies produced from all the tested isolates showed irregular shape, black microsclerotia, and hyaline, elliptical, single-celled conidia developed on verticillate conidiophores (2). For molecular identification, two specific primer pairs (Ver2-Ver3 and Vd7b-Vd10) of the intergenic spacer region (IGS) were used (3). A fragment of 339 bp was obtained by Ver2-Ver3 primer pair, which is a genus-specific primer, while a fragment of 139 bp was amplified by Vd7b-Vd10 primer pair specific for V. dahliae. To fulfil Kochs postulates, 10-month-old mango plants cv. Kensington Pride were inoculated by dipping the roots in Verticillium conidial suspension for 10 min. Conidial suspension was approximately 4 × 106 conidia/ml, prepared from Verticillium isolates Vd-1 and Vd-2 (10 plants for each isolate). Plants dipped in sterile water were used as controls. All plants were kept in a greenhouse at 25°C and 90 to 95% relative humidity. After 10 months, all inoculated plants showed symptoms identical to those observed in mango plants in the field; plants inoculated with water did not show symptoms. V. dahliae was consistently isolated from symptomatic plants but not from plants inoculated with water. To our knowledge, this is the first report of Verticillium wilt caused by V. dahliae on mango in Italy. Verticillium wilt is known to be a serious threat for the mango industry worldwide. The disease, still localized in Catania Province, may soon affects all Sicilian mango-growing provinces with serious economic consequences. References: (1) Y. M. Ahmed et al. J. Plant Pathol. 92:S4.71, 2010. (2) D. L. Hawksworth and P. W. Talboys. CMI Descriptions of Pathogenic Fungi and Bacteria, No. 256. CAB International, Wallingford, UK, 1970. (3) L. Schena et al. Phytopathol. Mediterr. 43:273, 2004.

Effect of potassium sorbate (E-202) and the antifungal PgAFP protein on Aspergillus carbonarius growth and ochratoxin A production in raisin simulating media: Use of potassium sorbate and PgAFP protein to prevent ochratoxin A in raisins

BACKGROUND Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi. The presence of OTA in raisins is mainly related to black Aspergillus spp. contamination. This toxin poses risks to human and animal health due to its high toxicity and carcinogenicity. New strategies to avoid the risk associated with OTA are therefore necessary. RESULTS A comparison was made between the effects of the antifungal protein PgAFP and potassium sorbate (E-202) on the growth of Aspergillus carbonarius, biosynthetic- and stress-related gene expression and its OTA production at two water activity (aw ) levels, 0.95 and 0.93 aw . The results showed that PgAFP successfully controlled OTA production, whereas E-202, despite being able to reduce Aspergillus carbonarius growth, caused a significant increase in OTA production by the fungus. CONCLUSION PgAFP protein, a biological compound with an antifungal activity, is safer to use than E-202 and may be proposed as a food preservative and a useful strategy to control ochratoxigenic A. carbonarius in raisins.