Claudio M. G. Oliveira

Molecular and morphometric analyses of Xiphidorus species (Nematoda: Longidoridae)

Xiphidorus nematodes are indigenous to Latin America and have a restricted geographical distribution compared to Xiphinema. A principal component analysis (PCA) based on eight morphometric characters from 39 South American populations clearly separated populations previously identified as X. achalae, X. amazonensis, X. minor, X. saladillensis and X. uruguayensis and three undescribed Xiphidorus species. However, populations identified as X. balcarceanus, X. parthenus and X. yepesara did not form similar discrete groupings and exhibited either considerable morphological variability or were incorrectly identified. Maximum likelihood phylogenetic trees derived from both 18S rDNA and ITS-1 sequences discriminated six Xiphidorus species (X. balcarceanus, X. minor, X. parthenus, X. yepesara, and two undescribed Xiphidorus species) from Brazil. Also, restriction analysis of PCR products derived from the ITS-1 region using three restriction enzymes (Taq I, Rsa I and Hin f I) yielded repeatable patterns that clearly discriminated these six Xiphidorus species. Sequence divergence was noted between X. parthenus and X. yepesara. Our morphometric and molecular data suggests that X. parthenus and X. yepesara are distinct species contrary to their previous subspecific status.

Morphological and molecular characterisation of Aphelenchoides besseyi and A. fujianensis (Nematoda: Aphelenchoididae) from rice and forage grass seeds in Brazil

Morphologically similar Aphelenchoides spp. populations extracted from rice and forage grass seeds from different geographical regions in Brazil were morphologically and molecularly characterised. Overall, the populations studied separated into two groups based on morphological and phylogenetic analyses, referred to herein as ‘Group-rice’ and ‘Group-forage’. Bayesian phylogenetic analyses of SSU, LSU and mtCOI regions strongly supported the presence of two dichotomous groups with Group-rice and Group-forage populations genetically similar to A. besseyi and A. fujianensis , respectively. This study reports the presence of a morphologically similar species to A. besseyi associated with seeds of grasses, but genetically distinct based on three genomic regions, which our results strongly suggest to be A. fujianensis , this being a new geographical record for Brazil. Additional information regarding spicule morphology of male A. besseyi is also reported.

Coffee-Associated Pratylenchus spp. – Ecology and Interactions with Plants

This chapter focuses on the basic biology of coffee-parasitic Pratylenchus spp., and on their interaction with coffee plants at the cellular, tissue and physio-logical levels. The parthenogenic species P. brachyurus and the amphimitic P. coffeae are well adapted to tropical climates, being prevalent in Indian and Central American coffee plantations, while the former is prevalent in Brazil. Soil temperatures lower than 10°C and higher than 32°C, and soil moisture content below 2% are unfavorable to the survival of these species. Their survival in fallowing soil is less than four months, although they survive for at least nine months in decaying roots; alternate hosts are also important for these species’ epidemiology. It seems that edaphic conditions do not play a role in the distribution of Pratylenchus spp. on coffee. Both Pratylenchus species cause extensive damage in coffee root tissues, particularly in Coffea arabica; consequently, water and nutrient uptakes, photosynthesis and downward transport of sucrose are reduced; these processes originate the symptoms observed in parasitized coffee plants: stunting, severe chlorosis and leaf shedding.

Hydrolysis probe-based PCR for detection of Pratylenchus crenatus , P. neglectus and P. penetrans

Molecular detection of pest and pathogens relies on rapid and dependable methods for their identification as well as an assessment of their abundance. This study describes the development and evaluation of a diagnostic method for detection of Pratylenchus crenatus , P. penetrans and P. neglectus , based on a hydrolysis probe qPCR assay. Primer/probe sets were designed targeting the ITS-1 rDNA. In order to assess the specificity, primer/probe sets were tested with samples of non-target Pratylenchus species and Radopholus similis . Experiments using dilutions of purified plasmid standards tested the sensitivity of the hydrolysis assay against detection of DNA extracted from individual nematodes. Target DNA was detected in soil samples collected from potato fields and this indicated that P. crenatus , P. neglectus and P. penetrans are widely distributed in Scotland, frequently co-existing in mixed populations, with P. crenatus more prevalent than either P. neglectus or P. penetrans .

A Rapid Diagnostic for Detection of Aphelenchoides besseyi and A. fujianensis Based on Real-Time PCR

Aphelenchoides besseyi and A. fujianensis have been frequently found in mixed populations associated with forage grass seed in Brazil. The morphological similarity between both species has previously led A. fujianensis to be erroneously identified as A. besseyi. A. besseyi is a quarantine pest in many countries that import Brazilian forage seed; however, there is no current evidence suggesting that A. fujianensis is a plant-parasitic species. Two real-time polymerase chain reaction (qPCR) diagnostics were developed to detect each species and an operational envelope was established. A set of primers and hydrolysis probes for each species was designed targeting the large subunit (LSU) region. To assess their specificity, primers and probes sets were tested with samples of nontarget Aphelenchoides and Paraphelenchus sp. also frequently associated with forage seed. Experiments using dilutions of purified plasmid standards underpinned the sensitivity of the qPCR assays, which detected as few as 10 copies of target nematode ribosomal DNA. Thus, the developed diagnostics were sufficiently sensitive to detect DNA extracted from a fragment of a single target nematode. There was a positive correlation between copy number of the target species and nematode abundance, suggesting the potential of this method for quantification. Evidence of intra-individual variability among cloned sequences of the LSU region in a single A. besseyi population is also reported.