Crop rotation with Meloidogyne-resistant germplasm is useful to manage and revert the (a)virulent populations of Mi1.2 gene and reduce yield losses.

A rotation sequence of ungrafted and grafted tomato-melon-pepper-watermelon on resistant rootstocks 'Brigeor', Cucumis metuliferus, 'Oscos' and Citrullus amarus, respectively, was carried out in a plastic greenhouse, ending with a susceptible or resistant tomato crop. The rotation was conducted in plots infested by an avirulent (Avi) or a partially virulent (Vi) Meloidogyne incognita population to the Mi1.2 gene. At the beginning of the study, the reproduction index (RI, relative reproduction in the resistant respect susceptible tomato) of Avi and Vi populations was 1.3% and 21.6%, respectively. Soil nematode density at transplanting (Pi) and at the end (Pf) of each crop, disease severity and crop yield were determined. Moreover, the putative virulence selection and fitness cost were determined at the end of each crop in pot tests. In addition, a histopathological study was carried out 15 days after nematode inoculation in pot test. The volume and number of nuclei per giant cell (GC) and the number of GC, their volume and the number of nuclei per feeding site in susceptible watermelon and pepper were compared with C. amarus and resistant pepper. At the beginning of the study, the Pi of Avi and Vi plots did not differ between susceptible and resistant germplasm. At the end of the rotation, the Pf of Avi was 1.2 the Pi in susceptible and 0.06 in resistant, the cumulative yield of grafted crops was 1.82 times higher than that of the ungrafted susceptible ones, and the RI in resistant tomato less than 10% irrespective of the rotation sequence. Concerning the Vi, Pf was below the detection level at the end of the rotation in resistant and 3 times Pi in the susceptible. The cumulative yield of grafted crops was 2.83 times higher than that of the ungrafted and the RI in resistant tomato was 7.6%, losing the population's virulence. In the histopathological study, no differences in number of GC per feeding site were observed in watermelon compared to C. amarus, but they were more voluminous and contained higher number of nuclei per GC and per feeding site. Regarding pepper, Avi population did not penetrate resistant rootstock.

Host Suitability of Lettuce and Bean Germplasm for Meloidogyne incognita and M. javanica Isolates from Spain.

Meloidogyne spp. are an important threat to horticulture and cause substantial yield losses. Plant resistance is an alternative control method for chemical nematicides. This study highlights the host suitability of the lettuces cultivars Grand Rapids and Salinas 88 and the beans cultivars Aporé, Cornell 49242, Macarrão Atibaia and Ouro Negro to four Meloidogyne incognita and seven M. javanica isolates from Spain in a pot experiment. Moreover, the response of these cultivars to increasing M. incognita densities (Pi) was assessed in a plastic greenhouse. The lettuce cultivar Regina 71 and the bean cultivar Bolinha were included as susceptible standards for comparison. It was found that Grand Rapids and Salinas 88 lettuces were resistant to the most nematode isolates in the pot experiment but were classified as slightly and moderately resistant, respectively, in the plastic greenhouse at increasing Pi. Regarding the beans, Aporé was resistant to the majority of the Meloidogyne isolates whereas Macarrão Atibaia and Ouro Negro were slightly resistant and Cornell 49242 was susceptible in the pot experiment. In the plastic greenhouse, Aporé was the only cultivar able to effectively suppress the nematode reproduction irrespective of Pi, while Ouro Negro became less resistant as Pi increased. These results play an important role in enhancing the effective and ecofriendly Meloidogyne management strategies.

Nasopharyngeal microbiota profiling of pregnant women with SARS-CoV-2 infection.

We aimed to analyze the nasopharyngeal microbiota profiles in pregnant women with and without SARS-CoV-2 infection, considered a vulnerable population during COVID-19 pandemic. Pregnant women were enrolled from a multicenter prospective population-based cohort during the first SARS-CoV-2 wave in Spain (March-June 2020 in Barcelona, Spain) in which the status of SARS-CoV-2 infection was determined by nasopharyngeal RT-PCR and antibodies in peripheral blood. Women were randomly selected for this cross-sectional study on microbiota. DNA was extracted from nasopharyngeal swab samples, and the V3-V4 region of the 16S rRNA of bacteria was amplified using region-specific primers. The differential abundance of taxa was tested, and alpha/beta diversity was evaluated. Among 76 women, 38 were classified as positive and 38 as negative for SARS-CoV-2 infection. All positive women were diagnosed by SARS-CoV-2 IgG and IgM/IgA antibodies, and 14 (37%) also had a positive RT-PCR. The overall composition of the nasopharyngeal microbiota differ in pregnant women with SARS-CoV-2 infection (positive SARS-CoV-2 antibodies), compared to those without the infection (negative SARS-CoV-2 antibodies) (p = 0.001), with a higher relative abundance of the Tenericutes and Bacteroidetes phyla and a higher abundance of the Prevotellaceae family. Infected women presented a different pattern of microbiota profiling due to beta diversity and higher richness (observed ASV < 0.001) and evenness (Shannon index < 0.001) at alpha diversity. These changes were also present in women after acute infection, as revealed by negative RT-PCR but positive SARS-CoV-2 antibodies, suggesting a potential association between SARS-CoV-2 infection and long-lasting shift in the nasopharyngeal microbiota. No significant differences were reported in mild vs. severe cases. This is the first study on nasopharyngeal microbiota during pregnancy. Pregnant women with SARS-CoV-2 infection had a different nasopharyngeal microbiota profile compared to negative cases.

Tomato and Melon Meloidogyne Resistant Rootstocks Improve Crop Yield but Melon Fruit Quality Is Influenced by the Cropping Season.

Four rotation sequences consisting of ungrafted tomato cv. Durinta - melon cv. Paloma or tomato grafted onto the resistant rootstock 'Aligator' - melon grafted onto the resistant Cucumis metuliferus accession BGV11135, and in reverse order, were conducted from 2015 to 2017 in a plastic greenhouse infested or not with Meloidogyne incognita to determine the plant tolerance (T), the minimum relative crop yield (m) and fruit quality. The relationship between M. incognita densities in soil at transplanting (Pi) of each crop and the crop yield was assessed and T and m were estimated by the Seinhorst's damage model. In addition, the volume and the number of nuclei of single giant cells and the number of giant cells, its volume and the number of nuclei per feeding site in susceptible tomato and melon were compared to those in the resistant tomato and C. metuliferus 15 days after nematode inoculation in pot test. The relationship between the Pi and the relative crop yield fitted the Seinhorst's damage model in both ungrafted and grafted tomato and melon, but not for all years and cropping seasons. The estimated T for ungrafted and grafted tomato did not differ but m was lower in the former (34%) than the latter (67%). Sodium concentration in fruits from ungrafted but not from grafted tomato increased with nematode densities in spring 2015 and 2016. The estimated ungrafted melon T did not differ from the grafted melon cultivated in spring, but it did when it was cultivated in summer. The relative crop yield of ungrafted melon was lower (2%) than the grafted cultivated in spring (62%) and summer (20%). Sodium concentration in melon fruits from ungrafted plants increased with nematode densities. No variations in fruit quality from grafted melon cultivated in spring were found, although less dry matter and soluble solid content at highest nematode densities were registered when it was cultivated in summer. Lower number of giant cells per feeding site was observed in both susceptible tomato germplasms compared to the resistant ones but they were more voluminous and held higher number of nuclei per giant cell and per feeding site.

Bacillus firmus Strain I-1582, a Nematode Antagonist by Itself and Through the Plant.

Bacillus firmus I-1582 is approved in Europe for the management of Meloidogyne on vegetable crops. However, little information about its modes of action and temperature requirements is available, despite the effect of these parameters in its efficacy. The cardinal temperatures for bacterial growth and biofilm formation were determined. The bacteria was transformed with GFP to study its effect on nematode eggs and root colonization of tomato (Solanum lycopersicum) and cucumber (Cucumis sativus) by laser-scanning confocal microscopy. Induction of plant resistance was determined in split-root experiments and the dynamic regulation of genes related to jasmonic acid (JA) and salicylic acid (SA) by RT-qPCR at three different times after nematode inoculation. The bacteria was able to grow and form biofilms between 15 and 45°C; it degraded egg-shells and colonized eggs; it colonized tomato roots more extensively than cucumber roots; it induced systemic resistance in tomato, but not in cucumber; SA and JA related genes were primed at different times after nematode inoculation in tomato, but only the SA-related gene was up-regulated at 7 days after nematode inoculation in cucumber. In conclusion, B. firmus I-1582 is active at a wide range of temperatures; its optimal growth temperature is 35°C; it is able to degrade Meloidogyne eggs, and to colonize plant roots, inducing systemic resistance in a plant dependent species manner.

Pochonia chlamydosporia Induces Plant-Dependent Systemic Resistance to Meloidogyne incognita.

Meloidogyne spp. are the most damaging plant parasitic nematodes for horticultural crops worldwide. Pochonia chlamydosporia is a fungal egg parasite of root-knot and cyst nematodes able to colonize the roots of several plant species and shown to induce plant defense mechanisms in fungal-plant interaction studies, and local resistance in fungal-nematode-plant interactions. This work demonstrates the differential ability of two out of five P. chlamydosporia isolates, M10.43.21 and M10.55.6, to induce systemic resistance against M. incognita in tomato but not in cucumber in split-root experiments. The M10.43.21 isolate reduced infection (32-43%), reproduction (44-59%), and female fecundity (14.7-27.6%), while the isolate M10.55.6 only reduced consistently nematode reproduction (35-47.5%) in the two experiments carried out. The isolate M10.43.21 induced the expression of the salicylic acid pathway (PR-1 gene) in tomato roots 7 days after being inoculated with the fungal isolate and just after nematode inoculation, and at 7 and 42 days after nematode inoculation too. The jasmonate signaling pathway (Lox D gene) was also upregulated at 7 days after nematode inoculation. Thus, some isolates of P. chlamydosporia can induce systemic resistance against root-knot nematodes but this is plant species dependent.

Commercial Formulates of Trichoderma Induce Systemic Plant Resistance to Meloidogyne incognita in Tomato and the Effect Is Additive to That of the Mi-1.2 Resistance Gene.

Meloidogyne is the most damaging plant parasitic nematode genus affecting vegetable crops worldwide. The induction of plant defense mechanisms against Meloidogyne in tomato by some Trichoderma spp. strains has been proven in pot experiments, but there is no information for tomato bearing the Mi-1.2 resistance gene or for other important fruiting vegetable crops. Moreover, Trichoderma is mostly applied for managing fungal plant pathogens, but there is little information on its effect on nematode-antagonistic fungi naturally occurring in soils. Thus, several experiments were conducted to determine (i) the ability of two commercial formulates of Trichoderma asperellum (T34) and Trichoderma harzianum (T22) to induce systemic resistance in tomato and cucumber against an avirulent Meloidogyne incognita population in split-root experiments; (ii) the effect of combining T34 with tomato carrying the Mi-1.2 resistance gene to an avirulent M. incognita population in sterilized soil; and (iii) the effect of combining T34 with tomato carrying the Mi-1.2 resistance gene to a virulent M. incognita population in two suppressive soils in which Pochonia chlamydosporia is naturally present, and the effect of T34 on the level of P. chlamydosporia egg parasitism. Both Trichoderma formulates induced resistance to M. incognita in tomato but not in cucumber. In tomato, the number of egg masses and eggs per plant were reduced by 71 and 54% by T34, respectively. T22 reduced 48% of the number of eggs per plant but not the number of egg masses. T34 reduced the number of eggs per plant of the virulent M. incognita population in both resistant and susceptible tomato cultivars irrespective of the suppressive soil, and its effect was additive with the Mi-1.2 resistance gene. The percentage of fungal egg parasitism by P. chlamydosporia was not affected by the isolate T34 of T. asperellum.

Cucumis metuliferus reduces Meloidogyne incognita virulence against the Mi1.2 resistance gene in a tomato-melon rotation sequence.

BACKGROUND: Susceptible tomato cv. Durinta, ungrafted or grafted onto cv. Aligator resistant rootstock, both followed by the susceptible melon cv. Paloma, ungrafted or grafted onto Cucumis metuliferus BGV11135, and in the reverse order, were cultivated from 2015 to 2017 in the same plots in a plastic greenhouse, infested or not with Meloidogyne incognita. For each crop, soil nematode densities, galling index, number of eggs per plant and crop yield were determined. Virulence selection was evaluated in pot experiments. RESULTS: In the tomato-melon rotation, nematode densities increased progressively for the grafted tomato, being higher than for ungrafted plants at the end of the study; this was not the case in the melon-tomato rotation. Grafted crops yielded more than ungrafted crops in the infested plots. Virulence against the Mi1.2 gene was detected, but not against C. metuliferus. Reproduction of M. incognita on the resistant tomato was ∼ 120% that on the susceptible cultivar after the first grafted tomato crop, but this decreased to just 25% at the end of the experiment. CONCLUSION: Alternating different resistant plant species suppresses nematode population growth rate and yield losses. Although this strategy does not prevent virulence selection, the level was reduced. © 2018 Society of Chemical Industry.

Induction of auxin biosynthesis and WOX5 repression mediate changes in root development in Arabidopsis exposed to chitosan.

Chitosan is a natural polymer with applications in agriculture, which causes plasma membrane permeabilisation and induction of intracellular reactive oxygen species (ROS) in plants. Chitosan has been mostly applied in the phylloplane to control plant diseases and to enhance plant defences, but has also been considered for controlling root pests. However, the effect of chitosan on roots is virtually unknown. In this work, we show that chitosan interfered with auxin homeostasis in Arabidopsis roots, promoting a 2-3 fold accumulation of indole acetic acid (IAA). We observed chitosan dose-dependent alterations of auxin synthesis, transport and signalling in Arabidopsis roots. As a consequence, high doses of chitosan reduce WOX5 expression in the root apical meristem and arrest root growth. Chitosan also propitiates accumulation of salicylic (SA) and jasmonic (JA) acids in Arabidopsis roots by induction of genes involved in their biosynthesis and signalling. In addition, high-dose chitosan irrigation of tomato and barley plants also arrests root development. Tomato root apices treated with chitosan showed isodiametric cells respect to rectangular cells in the controls. We found that chitosan causes strong alterations in root cell morphology. Our results highlight the importance of considering chitosan dose during agronomical applications to the rhizosphere.

Chitosan Increases Tomato Root Colonization by Pochonia chlamydosporia and Their Combination Reduces Root-Knot Nematode Damage.

The use of biological control agents could be a non-chemical alternative for management of Meloidogyne spp. [root-knot nematodes (RKN)], the most damaging plant-parasitic nematodes for horticultural crops worldwide. Pochonia chlamydosporia is a fungal parasite of RKN eggs that can colonize endophytically roots of several cultivated plant species, but in field applications the fungus shows a low persistence and efficiency in RKN management. The combined use of P. chlamydosporia with an enhancer could help its ability to develop in soil and colonize roots, thereby increasing its efficiency against nematodes. Previous work has shown that chitosan enhances P. chlamydosporia sporulation and production of extracellular enzymes, as well as nematode egg parasitism in laboratory bioassays. This work shows that chitosan at low concentrations (up to 0.1 mg ml-1) do not affect the viability and germination of P. chlamydosporia chlamydospores and improves mycelial growth respect to treatments without chitosan. Tomato plants irrigated with chitosan (same dose limit) increased root weight and length after 30 days. Chitosan irrigation increased dry shoot and fresh root weight of tomato plants inoculated with Meloidogyne javanica, root length when they were inoculated with P. chlamydosporia, and dry shoot weight of plants inoculated with both P. chlamydosporia and M. javanica. Chitosan irrigation significantly enhanced root colonization by P. chlamydosporia, but neither nematode infection per plant nor fungal egg parasitism was affected. Tomato plants cultivated in a mid-suppressive (29.3 ± 4.7% RKN egg infection) non-sterilized clay loam soil and irrigated with chitosan had enhanced shoot growth, reduced RKN multiplication, and disease severity. Chitosan irrigation in a highly suppressive (73.7 ± 2.6% RKN egg infection) sterilized-sandy loam soil reduced RKN multiplication in tomato. However, chitosan did not affect disease severity or plant growth irrespective of soil sterilization. Chitosan, at an adequate dose, can be a potential tool for sustainable management of RKN.

Arabidopsis thaliana root colonization by the nematophagous fungus Pochonia chlamydosporia is modulated by jasmonate signaling and leads to accelerated flowering and improved yield.

Pochonia chlamydosporia has been intensively studied in nematode control of different crops. We have investigated the interaction between P. chlamydosporia and the model system Arabidopsis thaliana under laboratory conditions in the absence of nematodes. This study demonstrates that P. chlamydosporia colonizes A. thaliana. Root colonization monitored with green fluorescent protein-tagged P. chlamydosporia and quantitative PCR (qPCR) quantitation methods revealed root cell invasion. Fungal inoculation reduced flowering time and stimulated plant growth, as determined by total FW increase, faster development of inflorescences and siliques, and a higher yield in terms of seed production per plant. Precocious flowering was associated with significant expression changes in key flowering-time genes. In addition, we also provided molecular and genetic evidence that point towards jasmonate signaling as an important factor to modulate progression of plant colonization by the fungus. Our results indicate that P. chlamydosporia provides benefits to the plant in addition to its nematophagous activity. This report highlights the potential of P. chlamydosporia to improve yield in economically important crops.

CAZyme content of Pochonia chlamydosporia reflects that chitin and chitosan modification are involved in nematode parasitism.

Pochonia chlamydosporia is a soil fungus with a multitrophic lifestyle combining endophytic and saprophytic behaviors, in addition to a nematophagous activity directed against eggs of root-knot and other plant parasitic nematodes. The carbohydrate-active enzymes encoded by the genome of P. chlamydosporia suggest that the endophytic and saprophytic lifestyles make use of a plant cell wall polysaccharide degradation machinery that can target cellulose, xylan and, to a lesser extent, pectin. This enzymatic machinery is completed by a chitin breakdown system that involves not only chitinases, but also chitin deacetylases and a large number of chitosanases. P. chlamydosporia can degrade and grow on chitin and is particularly efficient on chitosan. The relevance of chitosan breakdown during nematode egg infection is supported by the immunolocalization of chitosan in Meloidogyne javanica eggs infected by P. chlamydosporia and by the fact that the fungus expresses chitosanase and chitin deacetylase genes during egg infection. This suggests that these enzymes are important for the nematophagous activity of the fungus and they are targets for improving the capabilities of P. chlamydosporia as a biocontrol agent in agriculture.

Chitosan enhances parasitism of Meloidogyne javanica eggs by the nematophagous fungus Pochonia chlamydosporia.

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol.