Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

Species-specific DNA markers for identification of two root-knot nematodes of coffee: Meloidogyne arabicida and M. izalcoensis

Seven root-knot nematodes (RKN), including Meloidogyne exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida, M. izalcoensis and M. arenaria are major pathogens of coffee crop in the Americas. Species-specific primers for their identification have been developed for five of them and constitute a fast and reliable method of identification. Here we report a PCR-based assay for specific detection of M. arabicida and M. izalcoensis. Random Amplified Polymorphic DNA fragments specific for these two species were converted into sequence characterized amplified region (SCAR) markers. PCR amplification using the SCAR primers produced a specific fragment of 300 bp and 670 bp for M. arabicida and M. izalcoensis, respectively, which were absent in other coffee-associated Meloidogyne spp. tested. SCAR primers also allowed successful amplification of DNA from single second-stage juveniles (J2), males and females. In addition, these primers were able to unambiguously detect the target species in nematode suspensions extracted from soil and roots samples, in different isolates of the same species or when used in multiplex PCR reactions containing mixtures of species. These results demonstrated the effectiveness of these SCAR markers and their multiplex use with those previously developed for M. exigua, M. incognita, M. paranaensis, M. enterolobii and M. arenaria constitute an essential detection tool. This diagnostic kit will contribute for specific J2 identification of the major RKN infecting coffee from field samples in the Americas.     

Genetic diversity of Meloidogyne spp. from rice and identification of multiresistant sources in Oryza spp. accessions

Recently a Meloidogyne species complex was detected parasitizing and causing damage to irrigated rice in southern Brazil, highlighting the need to study the genetic diversity of these species and their pathogenicity to Oryza spp. in order to select genotypes of rice with multiple resistance. This study compared the genetic diversity of Brazilian Meloidogyne spp. isolates from irrigated rice and evaluated the reaction of four wild accessions of Oryza species (O. glumaepatula, O. longistaminata, O. grandiglumis, and O. alta) and two cultivated species, O. glaberrima and O. sativa (control) to M. ottersoni, M. oryzae, and two variants of M. graminicola (Est G2 and Est G3). Genetic variability was assessed using RAPD and AFLP markers. M. graminicola and M. ottersoni showed high intraspecific variability: 83.76% and 41.14%, respectively. Cluster analysis showed a clear separation among rice root-knot nematodes (RKNs) into subclades according to their esterase phenotypes with 100% bootstrap. For rice resistance screening, plants were inoculated with 5,000 eggs, and the nematode reproduction factor evaluated 90–120 days postinoculation. O. glumaepatula, an American wild species, was highly resistant or resistant to all rice RKNs tested and is a valuable source of multiple resistance. Overall, the other rice species also showed different levels of resistance. Conversely, O. longistaminata exhibited low levels of resistance. M. graminicola Est G3 was the most aggressive isolate. Sources of resistance against RKN in wild Oryza genotypes, especially in an AA genome like O. glumaepatula, may be of great interest for future breeding programmes in cultivated rice.     

Sewage sludge assessment on growth of Acacia mangium seedlings by principal components analysis and orthogonal contrasts

This experiment aimed to compare combinations of sewage sludge-based substrates on the growth of Acacia mangium seedlings by principal component analysis (PCA) and orthogonal contrasts (OC). It was conducted in structures of a forest nursery, localized in Southeast of Brazil, in 280 cm³ tubes constituted by 14 treatments and 5 replicates of four plants. When the seedlings reached 100 days after sowing, the treatments containing sewage sludge associated with vermiculite were those that stood out for the growth of A. mangium seedlings, showing the highest averages for the biometric features. The addition of coconut fiber and coffee straw did not produce good growth as well as the evaluated commercial substrate. It can be stated that the use of principal components and OC were efficient in evaluating the best substrate added to the sewage sludge having a limitation in identifying the best ratio between constituents of the substrate.     

Resistance to <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> expresses a hypersensitive-like response in <Emphasis Type="Italic">Coffea arabica</Emphasis>

Root-knot nematodes (RKN) are obligate parasite species of the genus Meloidogyne that cause great losses in Arabica coffee (Coffea arabica L.) plantations. Identification of resistant genotypes would facilitate the improvement of coffee varieties aiming at an environmental friendly and costless nematode control. In this work, the C. arabica genotype ‘UFV 408-28’ was found to be resistant to the most destructive RKN species M. incognita. Pathogenicity assays indicated that the highly aggressive populations of M. incognita races 1, 2 and 3 were not able to successfully reproduce on ‘UFV 408-28’ roots and displayed a low gall index (GI = 2). An average reduction of 87% reduction of the M. incognita population was observed on ‘UFV 408-28’ when compared to the susceptible cultivar ‘IAC 15’. By contrast, ‘UFV 408-28’ was susceptible to the related species M. exigua and M. paranaensis (GI = 5 and 4, respectively). Histological observations performed on sections of UFV408-28 roots infected with M. incognita race 1 showed that nematode infection could be blocked right after penetration or during migration and establishment stages, at 6 days, 7 days and 8 days after infection (DAI). Fluorescence and bright field microscopy observations showed that root cells surrounding the nematodes exhibited HR-like features such as accumulation of phenolic compounds and a necrotic cell aspect. In the susceptible ‘IAC 15’ roots, 6 DAI, feeding sites contained giant cells with a dense cytoplasm. Necrotic cells were never observed throughout the entire infection cycle. The HR-like phenotype observed in the ‘UFV 408-28’—M. incognita interaction suggests that the coffee resistance may be mediated by a R-gene based immunity system and may therefore provide new insights for understanding the molecular basis of RKN resistance in perennial crops.     

Silicon mitigates boron deficiency and toxicity in cotton cultivated in nutrient solution

Silicon (Si) application, both via foliar application and via roots, may be promising to improve plant growth under different biotic or abiotic stresses. In the present study, we investigated whether application of Si can also mitigate the harmful effects of boron (B)‐related nutritional disorders, such as B deficiency, when the application of B is inefficient or insufficient, and B toxicity, when the soil presents high levels of B. This may enable producers to apply Si preventively, if there is a low availability of B in the environment or if B deficiency is induced during the growth season due to a water deficit reducing the plant's B absorption. The objective of this study was to investigate the influence of leaf and root Si application on alleviating the harmful effects of B deficiency and toxicity in cotton. Three experiments were carried out with cotton plants (Gossypium hirsutum cv. Bayer FM910®), using a soilless system. In a first experiment, we determined that highest plant Si concentrations were obtained with application of stabilized sodium and potassium silicate at concentrations of 0.8 g L^?1 (foliar) and 0.056 g L^?1 (roots). Experiment 2 indicated that the B concentrations in the nutrient solution associated with moderate B deficiency, sufficiency and moderate toxicity were 33.7, 83.6, and 130.5 µM B L^?1, respectively. In Experiment 3 we evaluated the effect of optimum Si applications on the physiology and dry weight production of cotton plants subjected to B deficiency, sufficiency, and toxicity. Silicon mitigated the harmful effects of both B deficiency and toxicity by increasing whole‐plant biomass production and levels of chlorophyll a, chlorophyll b, and total chlorophyll, and reduced initial and maximum fluorescence, thereby improving the quantum efficiency of photosystem II. Collectively, these results indicate that the greatest benefit of Si in mitigating B deficiency occurred with foliar B application, while Si supplied via the nutrient solution was more effective against B toxicity.     

A novel species-specific satellite DNA family in the invasive root-knot nematode <Emphasis Type="Italic">Meloidogyne mayaguensis</Emphasis> and its potential use for diagnostics

The root-knot nematode (RKN) Meloidogyne mayaguensis is considered as one of the most damaging RKN species because of its extremely wide host range. Recent surveys have shown the rapid spread of this parasite in agro-ecosytems, often making crop cultivation not viable in the heavily infested areas. Here, we report the identification, molecular cloning, genomic organisation and sequence analysis of a new satellite DNA (satDNA) family from M. mayaguensis (named pMmPet). It is comprised of two groups of A+T rich, tandemly repeated units of 174 and 180 bp, respectively. Using these sequences as targets, hybridisation and PCR experiments performed on a wide collection of 44 populations belonging to 15 RKN species showed that the pMmPet family could only be detected in the 16 M. mayaguensis populations tested. In addition, because of their repetitive nature, positive detection of pMmPet sequences was achieved in single individual nematodes. Therefore, the repeated sequence described here possesses features that make it an excellent candidate for use as a specific and extremely sensitive tool for the accurate detection and identification of this invasive pest on a routine basis. Clearly, monitoring the occurrence and spread of M. mayaguensis at the domestic and international levels are needed to avoid wholesale loss of agricultural resources in the infested regions.