Selection of Pinus spp. in South Africa for tolerance to infection by the pitch canker fungus

The increasing threats from pests and diseases demand that the South African forest industry explores options to deploy alternative pine species in plantation development. This is especially true for species, such as Pinus patula Schiede and Deppe ex Schltdl. and Cham., which are highly susceptible to the pitch canker fungus Fusarium circinatum. Losses due to F. circinatum have been confined mostly to nurseries and at field establishment resulting in a significant cost to the industry. Although, the fungus has not as yet resulted in stem and branch infections on established P. patula in South Africa, it has caused pitch canker on other, more susceptible species such as P. radiata D. Don., and P. greggii Engelm. ex Parl. As alternatives to P. patula, on the warmer and cooler sites in South Africa, families of P. elliottii Engelm var. elliottii, P. tecunumanii (Schw.) Eguiluz and Perry, P. maximinoi H. E. Moore and P. pseudostrobus Lindl. were screened for tolerance to infection by F. circinatum in greenhouse studies. Seedlings were wounded and inoculated with spores of F. circinatum. Lesion development following inoculation was used to differentiate the levels of tolerance between families. The results showed that P. maximinoi, P. pseudostrobus, and the low elevation variety of P. tecunumanii are highly tolerant to infection with very little family variation. The narrow sense heritability estimates for these species were less than 0.06. In contrast, P. elliottii showed good tolerance with some family variation and a heritability of 0.22, while the high elevation source of P. tecunumanii showed a high degree of family variation and a heritability of 0.59. These results provide the industry with valuable information on pine species tolerant to F. circinatum that could be used as alternatives to P. patula in South Africa.     

Carbon fractions as indicators of organic matter dynamics in chestnut orchards under different soil management practices

Several studies have emphasized the negative impact of the conventional soil management (CT) system on productivity and sustainability of chestnut orchards (Castanea sativa Mill.) when compared to no-tillage with grass cover (NT). However, scarce information is available regarding the effects of these soil management systems on soil organic matter (SOM) dynamics and soil quality. SOM or soil organic carbon is a key component of soil quality and has different fractions with different lability, namely, organic C (POC), active C (AC) and hot-water extractable carbon (HWC). These are considered as indicators of changes in management-induced soil quality. Thus, a study was carried out to evaluate the effects of NT and CT systems applied in the chestnut orchards on: (i) total amount of soil organic C (TOC), including C from both organic and mineral layers; (ii) soil organic C concentration of mineral horizons (OC); (iii) labile soil organic fractions (POC, AC, HWC); (iv) and soil mineral-associated C. The study was developed in two 30-year old chestnut orchards located in Northeast Portugal, that were kept under different soil management systems (NT or CT) during the preceding 17 years. Soil samples were taken at 0–10 and 10–20 cm soil depth. No significant differences in OC concentration were observed between NT and CT, while TOC was significantly higher in NT than in CT (22.54 and 12.17 Mg/ha or 34.16 and 22.90 Mg/ha, considering the organic layer plus mineral layers at 0–10 and 0–20 cm depth (set of two depths). The NT practice led to significantly higher concentration of labile C fractions (POC, AC and HWC) than CT at 0–10 cm soil depth. These results indicate that measurement of labile soil organic C fractions, such as POC, AC and HWC, may provide a sensitive and consistent indication of changes in soil C and SOM dynamics in response to soil management practices. Overall, NT seems to ensure better soil quality than CT in chestnut orchards under Mediterranean climate conditions.     

Low Concentrations of Glyphosate-Based Herbicide Affects the Development of <Emphasis Type="Italic">Chironomus xanthus</Emphasis>

Glyphosate is an herbicide commonly used worldwide for weed control and generally applied as part of a formulated product, such as Roundup. Contamination of surface water by glyphosate-based herbicides can cause deleterious effects in organisms, mainly in aquatic systems near to intensive agricultural areas (e.g., transgenic soybean crops). Given the lack of toxicological information concerning effects of glyphosate-based herbicides on tropical aquatic ecosystems, we aimed to evaluate the lethal and sub-lethal effects of Roundup Original® on the dipteran Chironomus xanthus. The endpoints evaluated included survival, growth, and emergence. The results showed that the 48 h LC₅₀ for glyphosate to C. xanthus was 251.5 mg a.e./L. Larval growth of C. xanthus was reduced under glyphosate exposure (LOEC for body length = 12.06 mg/L; LOEC for head capsule width = 0.49 mg/L). No effects were observed in terms of cumulative percentage of imagoes emergence. However, low concentrations of glyphosate caused delayed emergence of females (at 1.53 mg/L) and induced fast emergence of males (at 0.49 mg/L), compared to control treatment. The deleterious effects of environmental relevant concentrations of glyphosate (0.7 mg/L) observed in terms of C. xanthus growth and development suggest that glyphosate-based herbicides can have negative consequences for aquatic non-target invertebrates such as Chironomus. Multigerational assays are needed to assess the long term effects of glyphosate on C. xanthus populations. Finally, our study adds ecotoxicological data on the effects of glyphosate-based herbicides on tropical freshwater invertebrates.     

Discriminating Organic and Conventional Coffee Production Systems Through Soil and Foliar Analysis Using Multivariate Approach

This study tested the hypothesis that evaluating soil and leaf nutrient contents is useful to discriminate organic and conventional management systems of coffee plantations. The study consisted of areas planted with Coffea arabica under organic or conventional management located in Espírito Santo, Brazil. We detected significant differences between both management systems when we utilized multivariate statistics to discriminate the areas. In our experiments, the principal mineral leaf nutrients, which acted as indicators were phosphor (P), calcium (Ca), magnesium (Mg), sodium (Na), zinc (Zn), and manganese (Mn), with higher contents in the organic coffee production system. The only mineral nutrient, which showed significantly lower values in leaves as well as in soil of organic coffee is boron (B). The implications of these findings are discussed. However, when using univariate statistics, like ANOVA or t-test, we did not find any significant difference between both management systems, although using the same dataset. Therefore, to discriminate between complex systems, we always recommend to recur to multivariate methodologies that are more adequate for such cases.     

Effect of time of oocyte collection and site of insemination on oocyte transfer in mares

The objective of the study was to compare embryo development rates after transfer of oocytes collected 22 or 33 h after hCG injection into recipients inseminated within the uterus or the oviduct. Oocytes were collected at approximately 22 or 33 h after hCG injections and incubated for approximately 16 or 1.5 h, respectively, before transfer. Intrauterine inseminations using 1 x 10(9) progressively motile sperm were done approximately 12 h before and 2 h after transfer. For intraoviductal inseminations (gamete intrafallopian transfer [GIFT]), semen was centrifuged through a Percoll gradient, and 200,000 progressively motile sperm were transferred with oocytes into the oviduct. Time of oocyte collection (22 or 33 h) after hCG injection did not affect embryo development rates (17/25, 68%, vs 12/23, 52%, respectively; P = 0.40). When results from oocyte collections at 22 and 33 h after hCG were combined, oocyte transfer with intraoviductal vs intrauterine insemination resulted in similar (P = 0.70) embryo development rates (12/22, 55%, and 17/26, 65%, respectively). However, the interaction between time of oocyte collection and site of insemination tended to be significant (P = 0.09), suggesting that GIFT using oocytes collected at 33 h after hCG may not be as effective as using oocytes collected at 22 h after hCG. Because intraoviductal insemination requires a low number of sperm, GIFT could be used in cases of male subfertility, frozen semen, or sexed sperm.     

Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato

Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.     

Evaluation of ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid for tolerance to Xanthomonas axonopodis pv. citri and Xylella fastidiosa

Asiatic citrus canker (ACC), caused by Xanthomonas axonopodis Starr & Garces pv. citri (Hasse) Vauterin et al., and citrus variegated chlorosis (CVC), caused by Xylella fastidiosa Wells et al., are considered the main diseases affecting sweet orange scion varieties in Brazil. Among commercial varieties, mandarins and tangerines are recognized as tolerant to these pathogens. We report herein the production of ‘Hamlin’ sweet orange (Citrus sinensis L. Osbeck) + ‘Montenegrina’ mandarin (Citrus deliciosa Ten.) allotetraploid somatic hybrid plants by protoplast fusion with improved disease tolerance that could be used as a donor of resistance genes in interploid hybridisation. Somatic hybridisation was confirmed by leaf morphology, flow cytometry and RAPD analyses. The somatic hybrid was propagated by grafting and cultivated in a screenhouse for tolerance assays. For X. axonopodis pv. citri assays, buds were collected from both ‘Hamlin’ sweet orange and the somatic hybrid and grafted onto ‘Cleopatra’ mandarin (Citrus reshni hort. ex Tanaka). As a negative control, buds from ‘Mexerica Tardia’ mandarin (C. deliciosa) were collected and grafted onto ‘Cleopatra’ mandarin. Two-month old plants with at least one young vegetative flush were individually spray-inoculated with a 10⁶ CFU mL⁻¹X. axonopodis pv. citri suspension and incubated in a growth chamber, at 27 °C, under 16-h photoperiod. The somatic hybrid showed a statistically significant reduction in susceptibility to ACC 30 days after inoculation. Compared to ‘Hamlin’ sweet orange, disease severity was reduced by 70%, with similar tolerance to that of the mandarin negative control. For X. fastidiosa assays, buds were collected from the somatic hybrid and its parental plants and grafted onto ‘Rangpur’ lime (Citrus limonia Osbeck). The developed plants were needle-inoculated with a X. fastidiosa suspension (8.7 × 10¹⁰ CFU mL⁻¹) into the new growth flush stem. Bacterial population was quantified both at 4 (at the inoculation point) and 8 months (50 cm above the inoculation point) after inoculation. The first evaluation detected X. fastidiosa in 63% of ‘Hamlin’ sweet orange and ‘Hamlin’ + ‘Montenegrina’ mandarin samples. In the second evaluation, X. fastidiosa was detected in 47.4% of ‘Hamlin’ sweet orange and 10.5% of ‘Hamlin’ + ‘Montenegrina’ somatic hybrid samples, suggesting that bacterial movement was restricted in the somatic hybrid. X. fastidiosa was not detected in both evaluations in samples collected from leaves of ‘Montenegrina’ mandarin. These results indicate that the ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid has potential for improved disease tolerance that should enhance its value regarding future use in citrus breeding programs.     

Field study on nematode resistance in Nelore-breed cattle

The present study evaluated Nelore cattle with different degrees of resistance to natural infections by gastrointestinal nematodes. One hundred weaned male cattle, 11-12 months of age, were kept on the same pasture and evaluated from October 2003 to February 2004. Faecal and blood samples were collected for parasitological, haematological and immunological tests. In February 2004, the 10 most resistant and the 10 most susceptible animals were selected based on individual means of nematode faecal egg counts (FEC). Such animals were slaughtered for worm burden determination and nematode species identification. The repeatability estimates for FEC (+/-S.D.), log-transformed FEC and packed-cell volume (PCV) in all animals were 0.3 (+/-0.05), 0.26 (+/-0.04) and 0.42 (+/-0.05), respectively. The resistant group showed lower FEC and worm burdens than the susceptible group (P<0.05). There were no significant differences between groups regarding mean body weight, weight gain, PCV and total serum protein values (P>0.05). The resistant group showed higher total serum IgE levels (P<0.05) and higher mean eosinophil blood counts. However, the latter was statistically significant only 42 days after the beginning of the study. Nematodes Cooperia punctata and Haemonchus placei were predominant and the correlation between Cooperia and Haemonchus burdens was 0.64 (P<0.05), which indicated that animals presenting increased numbers of one of those genera probably had increased numbers of the other. The current study provides further evidence of IgE active role in nematode immunity and suggests that total serum IgE level might serve as an additional marker to select Nelore cattle that are responsive to H. placei and C. punctata infections.     

Virulence markers and genetic relationships of Shiga toxin-producing Escherichia coli strains from serogroup O111 isolated from cattle

Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy cattle (O111:NM, seven strains; O111:H8, three strains) in Brazil were studied and compared to previously characterized human strains in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. Most bovine STEC O111 strains were isolated from dairy calves, and strains with genotypes stx1 alone and stx1/stx2 (variant stx2) occurred in different regions. Irrespective of the stx genotype, all strains were positive for eae theta, alpha variants of tir, espA and espB, and for ler, qseA, iha, astA and efa1 genes. Only one strain was negative for EHEC-hlyA and all strains were negative for iha, saa and espP genes and for EAF and bfpA, genetic markers of EPEC. Except for the presence of stx2, bovine strains showed the same profile of putative virulence genes found among the human strains. Similar biochemical behavior was identified among the strains analysed. Two bovine STEC strains produced the localized adherence (LA) phenotype in 6-h tests with Caco-2 (human enterocyte) cells. Intimate attachment (judged by the FAS test) was found in 9 out of 10 bovine strains as it was observed for the human STEC strains. RAPD-PCR analysis showed two distinct RAPD groups among the STEC O111 strains examined. Despite the relative low frequency of STEC O111 strains recovered from cattle no differences in their pathogenic potential were observed compared to some strains isolated from human diarrhea, suggesting that healthy cattle may be a potential source of infection for humans in Brazil.     

Isolation of Malassezia pachydermatis and M. sympodialis from the external ear canal of cats with and without otitis externa

The objective of this study was to determine the presence of Malassezia spp. in the external ear canal of cats with and without otitis. Forty-five animals were studied, 20 with and 25 without otitis externa (OE). Cerumen or secretion from external ear canal samples was cultured on modified Mycosel agar and sterile olive oil was added to the surface of the medium before specimen seeding. The isolates were analysed for macro- and micromorphology and identified by catalase tests and on the basis of growth on Tween 20, 40, 60 and 80. Malassezia spp. were isolated from 15 out of 20 (75%) animals with otitis and from 7 out of 25 (28%) cats without OE; the difference between the two groups was statistically significant (P < or = 0.05). Malassezia pachydermatis and M. sympodialis were isolated from 60% (12/20) and 40% (8/20) of cats with otitis, respectively, with no significant difference in the frequency of isolation between the two species. In the microflora of the healthy ear canal M. pachydermatis was significantly more common (6/25, 24%) than M sympodialis (1/25, 4%). The present investigation confirms that M. sympodialis can also act as an aetiological agent of feline OE, and if commercial veterinary laboratories do not use media with added lipids for the isolation of Malassezia spp., this might lead to false-negative results.