William Mário de Carvalho Nunes

Assessment of genetic variability in grapefruits (Citrus paradisi Macf.) and pummelos (C. maxima (Burm.) Merr.) using RAPD and SSR markers

The genetic variability of 38 grapefruit (Citrus paradisi Macf.) and three pummelos (C. maxima (Burm.) Merr..) accessions was evaluated using RAPD, and single sequence repeat (SSR) analyses. Approximately49% of the 198 RAPD were polymorphic, and 4.6 alleles per SSR loci were identified. PIC values changed from 0.093 to 0.450. A UPGMA phenetic tree was constructed and two main grapefruit groups were identified. The grapefruit accessions `do Cabo and `Siamesa-Filipinasclustered very close to the pummelos in Group A. The Group B consisted of three sub-groups, which comprised all of the other grapefruit accessions. The majority of grapefruit accessions showed a narrow genetic base suggesting that the observed morphological polymorphism within the group must be associated with somatic mutations, which were not detected by these molecular markers.

Stability of Citrus tristeza virus protective isolates in field conditions

The objective of this work was to monitor the maintenance of Citrus tristeza virus (CTV) protective isolates stability in selected clones of Pera sweet orange (Citrus sinensis), preimmunized or naturally infected by the virus, after successive clonal propagations. The work was carried out in field conditions in the north of Parana State, Brazil. Coat protein gene (CPG) analysis of 33 isolates collected from 16 clones of Pera sweet orange was performed using single strand conformational polymorphism (SSCP). Initially, the isolates were characterized by symptoms of stem pitting observed in clones. Then viral genome was extracted and used as template for the amplification of CPG by reverse transcription polimerase chain reaction (RTPCR). RTPCR products electrophoretic profiles were analyzed using the Jaccard coefficient and the UPGMA method. The majority of the clones had weak to moderate stem pitting symptoms and its CTV isolates showed alterations in the SSCP profiles. However, the stability of the protective complex has been maintained, except for isolates from two analised clones. Low genetic variability was observed within the isolates during the studied years.

Populational fluctuation of vectors of Xylella fastidiosa, wells in sweet orange [Citrus sinensis (L.) Osbeck] varieties of northwest Paraná State, Brazil

The aim of the present study was to assess the population flutuation of the sharpshooters species subfamily Cicadellinae belonging to the tribes Cicadellini and Proconiini, in sweet orange [Citrus sinensis( L). Osbeck] commercial orchards of the northwest region of Parana State , Brazil. Samplings were carried out the employing every time 24 yellow sticky cards. Identification of the species showed that the most representative were Dilobopterus costalimai of the Cicadellini tribe and Acrogonia citrina of the Proconiini tribe.

First Report of Leaf Spot Caused by Cladosporium perangustum on Syagrus oleracea in Brazil

Syagrus oleracea (Mart.) Becc. (bitter coconut), a palm tree species that is native to central Brazil, has been increasingly cultivated in this country for heart-of-palm production. Epidemics of a necrotic leaf spot of unknown etiology have been recorded on bitter coconut plants in transplant nurseries and plantation since 2008. The first symptoms appear as small, yellow, hydrotic flecks on young or mature leaves that evolve to necrotic brown streaks that run parallel to the leaf veins. Usually, yellow halos occur around the lesions and hydrosis is common during lesion expansion. Necrotic lesions can reach up to 40 mm in length and 10 mm in width, and the lesions often coalesce, causing extensive tissue damage. During a survey in a 3-year-old bitter coconut plantation in Maringá County (coordinates: 23°2351.25″ S, 51°5702.09″ W; elevation: 507 m) in the state of Parana, a dozen symptomatic leaves were collected with the aim of elucidating the etiology of this disease. Conidia and conidiophores typical of Cladosporium were frequently observed on the diseased leaf tissue under natural field conditions as well on the surfaces of disinfected leaf tissues kept in a humid chamber for 48 h at 25 ± 2°C with a 12-h photoperiod. Five monoconidial cultures growing on potato dextrose agar (PDA) medium were obtained from different leaves showing leaf spot symptoms. The cultures were grown on PDA to induce sporulation. At 7 days after incubation at 25 ± 2°C and a 12-h photoperiod, gray to gray-olive colonies were observed. The conidiophores were macronematous, erect, oblong, branched, 1 to 5 septate, and 75.0 to 120.0 × 1.90 to 3.20 μm. The ramoconidia were cylindrical or oblong, 0 to 2 septate, and 28.0 to 40.0 × 2.8 to 3.6 μm, with a truncate base of 1.9 to 2.2 μm; secondary ramoconidia were cylindrical or oblong, 0 to 2 septate, 8.0 to 31.0 × 2.2 to 3.1 μm, with 3 to 5 distal conidial hila; intercalary 1-septate conidia were 5.5 to 17.0 × 2.1 to 3.4 μm, with 1 to 3 distal conidial hila; terminal 1-septate conidia were catenulate and 2.2 to 4.2 × 1.8 to 3.1 μm. Species identification was performed based on morphology and DNA sequence data (1). Portions of the elongation factor 1α (551 bp; TEF) and actin (213 bp; ACT) genes were amplified by PCR. A BLAST search of the GenBank database revealed that the TEF (KC484658 to KC484662) and ACT (KC484663 to KC484667) sequence fragments from isolates Gua1, Gua2, Gua3, Gua4, and Gua5 had 100% identity with the accessions HM148616 and HM148371 of Cladosporium perangustum (1). Isolates were tested for pathogenicity against bitter coconut. Ten potted plants with 4 to 6 fully expanded leaves were inoculated with each isolate by spraying a suspension of conidia (105 spores per ml) onto leaves until runoff using a handheld spray bottle. Non-inoculated controls (10 plants) were sprayed with distilled water. The plants were kept in a humid plastic chamber at 20 to 26°C. All examined isolates were pathogenic to bitter coconut, causing symptoms identical to those described above 12 days after inoculation. All inoculated tissues were plated onto PDA to confirm the presence of the pathogen. Live cultures are being maintained at the Micoteca/URM/UFPE ( www.ufpe.br/micoteca ), Brazil. To our knowledge, this is the first report of a disease caused by C. perangustum on S. oleracea worldwide, and the study provides valuable plant disease diagnostic information for the palm hearth industry in Latin America. Reference: (1) K. Bensch et al. Stud Mycol. 67:1, 2010.

Development and Validation of Standard Area Diagrams as Assessment Aids for Estimating the Severity of Citrus Canker on Unripe Oranges

Asiatic citrus canker (ACC) is an important disease of citrus in Brazil and elsewhere in the world. Infection with the causal pathogen, Xanthomonas citri subsp. citri, can cause severe disease on the fruit. Visual estimation of severity is the usual method used to quantify ACC on diseased fruit. The objective of this research was to construct and validate standard area diagram (SAD) sets as assessment aids for raters to improve the accuracy and reliability of visual estimates of ACC on unripe (green) fruit of sweet orange. Two SAD sets were constructed. A five-diagram SAD set had five severities depicted (0.5, 2.0, 8.0, 27.0, and 40.0%) and a six-diagram SAD set had six severities depicted (0.5, 1.0, 3.0, 9.0, 20.0, and 40.0%). Fifteen raters evaluated 40 images of cankered, unripe fruit. Both the five- and six-diagram SAD sets significantly improved the accuracy and reliability of estimates. Agreement, measured by Lins concordance correlation coefficient, was 0.220 to 0.913 when not using SADs, 0.814 to 0.955 when using five-diagram SAD sets, and 0.863 to 0.925 when using six-diagram SAD sets. The five-diagram SAD set was significantly more accurate and reliable compared with the six-diagram set. Possible reasons for this are discussed. Based on the results, the five-diagram SAD set is preferable to use. Although the SAD set was developed for sweet orange, it doubtless has applicability to other citrus, including grapefruit. These SAD sets should be useful for research endeavors where accurate and reliable estimates of the severity of ACC are required.

Comparison of Citrus tristeza virus (CTV) isolates by RFLP analysis of the coat protein nucleotide sequences and by the severity of the symptoms

Plants of Pera sweet orange on Rangpur lime rootstocks, from orchards of the northwest and north of Parana state, Brazil, were evaluated for severity of symptoms and genetic diversity of Citrus tristeza virus. The severity of symptoms was evaluated by the development of tree, fruit size and stem pitting symptoms. Isolates that infect these plants were compared with known mild and severe isolates by analysis of restriction fragments length polymorphism (RFLP) of the coat protein nucleotide sequences (CPNS), amplified by the polymerase chain reaction (PCR) and undergone digestion with the restriction enzymes Hinf I and Rsa I. The severity of symptoms showed that the analyzed plants from the northwest orchards presented mild to moderate tristeza symptoms, while the plants from the north orchards exhibited moderate to severe symptoms. The RFLP analysis revealed that the CTV isolates are constituted by haplotype mixtures. Rsa I was the enzyme that best discriminated the genetic diversity among the analyzed isolates of CTV. Two main groups were generated by the UPGMA analysis. The isolates from the northwest orchards grouped with most of the mild isolates used as control, and a great part of the isolates from the north orchards, was correlated with the severe isolate Capao Bonito. Correlation between the stem pitting intensity and RFLP patterns, was demonstrated with some exceptions. The failure of protection of some isolates and the contamination of the rootstocks by the severe isolates, in field nursery conditions, before grafting with scions with mild isolates, were the hypotheses considered to explain the occurrence of severe CTV isolates in the North area of Parana State.

Avaliação da resistência de variedades de Citrus spp. à Xanthomonas citri subsp. citri na região Noroeste Paranaense, em condições de campo

Identification of cultivars with certain levels of resistance to citrus canker and desirable agronomic and commercial features is of great importance for the management of this disease. Evaluation of promising genotypes can be performed under controlled conditions or in the field. This study aimed to evaluate genotypes of sweet orange (Citrus sinensis), mandarin (Citrus reticulata) and hybrids for resistance to citrus canker at different times. The experiment was conducted in an experimental orchard in Maringa, Parana State. To determine the mean severity of the disease, diagrammatic scales were used to evaluate four plants per block, and branches in the middle portion of each plant were sampled. In addition, we calculated the Area Under the Disease Progress Curve (AUDPC). According to the obtained data, there were significant differences in the severity and AUDPC along the evaluations, and the severity varied from 0.25% to 4.88%, while AUDPC varied from 49.80 to 742.65. The citrus varieties Tang. Ponkan, Rosehaugh Nartjee, Harris, De wildt, Dekopon, Lee, South Africa, Tarocco#23, Solid Scarlet, Satsuma Okitsu SPA 29, Cristalina, Shamouti and Pera Olimpia had the lowest severity levels and were classified as resistant. Results suggest that these genotypes can be important tools in breeding programs of citrus genotypes for resistance to Xanthomonas citri subsp. citri.

Stability of Citrus tristeza virus protective isolate 'Pêra IAC' according to SSCP analysis of old and new lines of three sweet orange varieties

Clonal cleaning, followed by pre-immunization with protective complexes of Citrus tristeza virus(CTV), allowed the commercial cultivation of Pera sweet orange, a variety that has great importance for Brazilian citriculture but is sensitive to the virus. The use of mild protective isolates in other citrus varieties, even those more tolerant to CTV, can also be of interest to prevent the spread of severe isolates. The aim of this study was to characterize, by means of SSCP (Single Strand Conformational Polymorphism) analysis of the coat protein gene, CTV isolates present in plants of the sweet orange cultivars Pera, Hamlin and Valencia propagated from four budwood sources: 1) old lines, 2) nucellar lines, 3) shoot-tip-grafted lines, and 4) shoot-tip-grafted lines pre-immunized with the mild CTV protective isolate PIAC. We also evaluated the correlation of the obtained SSCP patterns to stem pitting intensity, tree vigor and fruit yield. SSCP results showed low genetic diversity among the isolates present in different trees of the same variety and same budwood source and, in some cases, in different budwood sources and varieties. Considering tristeza symptoms, lower intensity was noted for plants of new, shoot-tip-grafted and pre-immunized shoot-tip-grafted lines, compared to old lines of the three varieties. The observed SSCP patterns and symptomatology suggested that more severe CTV complexes infect the plants of old lines of all three varieties. The protective complex stability was observed in the SSCP patterns of CTV isolates of some shoot-tip-grafted and pre-immunized clones. It was concluded that the changes detected in other electrophoretic profiles of this treatment did not cause loss of the protective capacity of CTV isolate PIAC inoculated in the pre-immunization.

A standard area diagram set to aid estimation of the severity of Asiatic citrus canker on ripe sweet orange fruit

Asiatic citrus canker (ACC) is a major disease of citrus in many tropical and subtropical citrus-growing regions. Severe disease can develop on the fruit, making it unmarketable. Research activities need accurate and reliable methods to estimate the severity of citrus canker, most often done visually by raters. The objective was to validate and compare two standard area diagram sets (SADs) as aids for raters to improve the accuracy and reliability of visual estimates of canker severity on ripe fruit of sweet orange (unripe fruit are green, and have slightly different symptoms compared to ripe fruit, warranting different SADs). A 5-diagram SAD set (0.7, 2.0, 7.0, 21.0 and 39.0xa0%) and a 6-diagram SAD set (0.7, 2.0, 4.0, 10.0, 21.0 and 39.0xa0%) were compared; the 6-diagram set had an additional diagram in the most common severity range. Fifteen raters evaluated 40 images of cankered, unripe fruit. Both the 5- and 6-diagram SADs improved the accuracy and reliability of rater estimates. Agreement, measured by Lin’s concordance correlation coefficient (ρc) was 0.735 to 0.906 when not using SADs, 0.931 to 0.985 when using 5-diagram SADs, and 0.879 to 0.979 when using 6-diagram SADs. Mean inter-rater reliability (R2) was 0.638 when not using SADs, 0.889 when using 5-diagram SADs, and 0.834 when using 6-diagram SADs. The 5-diagram SADs resulted in significantly more accurate and reliable estimates compared with the 6-diagram set, and is thus recommended for use. The SADs should have application for estimating ACC severity on ripe fruit of other citrus, including grapefruit.

Espécies de Phytophthora associadas à gomose em pomares de citros no Estado do Paraná, Brasil

Citrus root rot is considered a disease of great importance to citriculture in Brazil and around the world. The etiology of this disease comprises a complex of Phytophthora species. Although citrus root rot is important, little is known about it in the producing regions of Parana State, Brazil. Therefore, this study aimed to identify Phytophthora species associated with root rot in citrus orchards in Parana. In the North and Northwest regions of this state and in Vale do Ribeira, samples were collected from the roots of plants showing symptoms of citrus root rot, as well as from the rhizosphere soil. In the laboratory, employing pear cv. Danjou as bait and the culture medium potato-dextrose-agar, 21 isolates of Phytophthora spp. were obtained. All isolates infected seedlings of Rangpur lime, reproducing the symptoms of root rot and presenting mycelial growth at 8 oC and 36 oC, except for the isolate PR20 at 36 oC. In vitro, these isolates were heterothallic, and 20 of them were compatible with the standard type A2, while one of them was compatible with the standard type A1. Twenty isolates formed papillate persistent sporangia of 25.5 - 62.0 µm length (C) and 27.9 - 49.6 µm width (L); the C/L ratio was 1.38:1. One isolate (PR20) had sporangia of 40.3 - 55.8 µm length and 27.9 - 37.2 width, forming papillale or bipapillate persistent sporangia of distorted forms and not forming chlamydospores. The optimum temperature for the growth of this isolate was between 20 and 28oC, while for the remaining isolates it was from 24 to 32oC; the latter had abundant production of globose chlamydospores with diameter ranging from 21.7 to 43.4 µm. According to the presented morphophysiological characteristics, of the 21 analyzed isolates, 20 belonged to the species P. nicotianae and one to the species P. citrophthora. Sequence analysis of genes of the rDNA ITS1-5.8S-ITS2 region, using the test Single-Strand Conformation Polymorphism (SSCP), confirmed P. nicotianae and P. citrophthora as the two Phytophthora species associated with root rot in citrus orchards in the state of Parana.