Francisco Javier Sorribas

A population of Meloidogyne javanica in spain virulent to the Mi resistance gene in tomato

A population of Meloidogyne javanica virulent to Mi-gene in tomato was identified in Spain. It reproduced similarly on resistant and susceptible tomato cultivars in the greenhouse, microplots, and in the field. In monoxenic cultures, reproduction of the virulent M. javanica was higher than that of an avirulent population on resistant but not on susceptible tomatoes. The virulent population suppressed tomato yield of both resistant and susceptible tomatoes by 29% in microplots. Initial population density (Pi) was inversely correlated with Pf (final population density)/Pi on both resistant and susceptible tomatoes in the field. A negative correlation was found between Pi and tomato yield for the susceptible but not for the resistant cultivar.

Effectiveness and profitability of the Mi-resistant tomatoes to control root-knot nematodes

Experiments were conducted to determine the effectiveness and profitability of the Mi-resistance gene in tomato in suppressing populations of Meloidogyne javanica in a plastic-house with a natural infestation of the nematode. Experiments were also conducted to test for virulence and durability of the resistance. Monika (Mi-gene resistant) and Durinta (susceptible) tomato cultivars were cropped for three consecutive seasons in non-fumigated or in soil fumigated with methyl bromide at 75 g m−2 and at a cost of 2.44 euros m−2. Nematode densities were determined at the beginning and end of each crop. Yield was assessed in eight plants per plot weekly for 6 weeks. The Pf/Pi values were 0.28 and 21.6 after three crops of resistant or susceptible cultivars, respectively. Growth of resistant as opposed to susceptible tomato cultivars in non-fumigated soil increased profits by 30,000 euros ha−1. The resistant Monika in non-fumigated soil yielded similarly (P > 0.05) to the susceptible Durinta in methyl bromide fumigated soil but the resistant tomato provided a benefit of 8800 euros ha−1 over the susceptible one because of the cost of fumigation. Selection for virulence did not occur, although the nematode population subjected to the resistant cultivar for three consecutive seasons produced four times more eggs than the population on the susceptible one. Such a difference was also shown when the resistant cultivar was subjected to high continuous inoculum pressure for 14 weeks. The Mi-resistance gene can be an effective and economic alternative to methyl bromide in plastic-houses infested with root-knot nematodes, but should be used in an integrated management context to preserve its durability and prevent the selection of virulent populations due to variability in isolate reproduction and environmental conditions.

Evaluation of a Native and Introduced Isolate of Pochonia chlamydosporia against Meloidogyne javanica

Growth chamber and plastic tunnel experiments were conducted to compare the ability of a native and introduced isolate of Pochonia chlamydosporia to colonize the rhizosphere of selected plant species and survive in soil. Effects of the isolates on population density of Meloidogyne javanica and yield of tomato after single or multiple fungal applications were also determined. In growth chamber experiments, both isolates showed a similar ability to colonise the rhizosphere of selected vegetables, except for the introduced isolate, which produced more colony forming units cm−2 of root surface on tomato and cabbage than the native one. In the tunnel house, both isolates parasitized eggs of M. javanica, and the native but not the introduced isolate increased parasitism after multiple applications. The native isolate was recovered more frequently from soil, and was a better colonizer of tomato roots than the introduced one irrespective of the number of fungal applications. Multiple fungal applications of either isolate reduced the nematode gall rating, and the native isolate also reduced the final egg population in roots. Neither isolates reduced final nematode densities in soil or affected tomato yield when compared to untreated plots.

Characterization of Soil Suppressiveness to Root-Knot Nematodes in Organic Horticulture in Plastic Greenhouse.

The fluctuation of Meloidogyne population density and the percentage of fungal egg parasitism were determined from July 2011 to July 2013 in two commercial organic vegetable production sites (M10.23 and M10.55) in plastic greenhouses, located in northeastern Spain, in order to know the level of soil suppressiveness. Fungal parasites were identified by molecular methods. In parallel, pot tests characterized the level of soil suppressiveness and the fungal species growing from the eggs. In addition, the egg parasitic ability of 10 fungal isolates per site was also assessed. The genetic profiles of fungal and bacterial populations from M10.23 and M10.55 soils were obtained by Denaturing Gradient Gel Electrophoresis (DGGE), and compared with a non-suppressive soil (M10.33). In M10.23, Meloidogyne population in soil decreased progressively throughout the rotation zucchini, tomato, and radish or spinach. The percentage of egg parasitism was 54.7% in zucchini crop, the only one in which eggs were detected. Pochonia chlamydosporia was the only fungal species isolated. In M10.55, nematode densities peaked at the end of the spring-summer crops (tomato, zucchini, and cucumber), but disease severity was lower than expected (0.2–6.3). The percentage of fungal egg parasitism ranged from 3 to 84.5% in these crops. The results in pot tests confirmed the suppressiveness of the M10.23 and M10.55 soils against Meloidogyne. The number of eggs per plant and the reproduction factor of the population were reduced (P < 0.05) in both non-sterilized soils compared to the sterilized ones after one nematode generation. P. chlamydosporia was the only fungus isolated from Meloidogyne eggs. In in vitro tests, P. chlamydosporia isolates were able to parasitize Meloidogyne eggs from 50 to 97% irrespective of the site. DGGE fingerprints revealed a high diversity in the microbial populations analyzed. Furthermore, both bacterial and fungal genetic patterns differentiated suppressive from non-suppressive soils, but the former showed a higher degree of similarity between both suppressive soils than the later.

Microbial parasites associated with Tylenchulus semipenetrans in citrus orchards of Catalonia, Spain

Abstract Citrus orchards in Catalonia, Spain were surveyed to identify microbial parasites of Tylenchulus semipenetrans, and their distribution and density. Of 62 orchards, 48 were positively infested with the citrus nematode. Fungal strains were isolated from single eggs, females or second-stage juveniles of the citrus nematode in 69% of the infested orchards. The fungi identified in order of occurrence were Paecilomyces lilacinus, Fusarium solani, Fusarium spp., Cylindrocarpon cylindroides, Verticillium fungicola, Cladosporium cladosporioides, F. oxysporum, Veronaea botryosa, Sepedonium chrysospermum, Volutella ciliata, Exophiala pisciphila and Acremonium sp. Fungal parasitism was related directly to the number of females g−1 of root and magnesium in the soil, and inversely, to the number of eggs g−1 of root and phosphorus in the soil (R 2=0.8654; P<0.0001). Nematode trapping fungi were isolated from soil samples in 29% of the orchards, and Monacrosporium elegans, Arthrobotrys dactyloides, A. javanica, A. superba and A. oligospora var. microspora, were the species present. Endospores of the hyperparasite Pasteuria adhering to vermiform stages of T. semipenetrans were found in 50% of the orchards infested with the citrus nematode.

Effect on resistance to Tylenchulus semipenetrans of hybrid citrus rootstocks subjected to continuous exposure to high population densities of the nematode

Experiments simulating interplanting of resistant rootstocks with susceptible rootstocks that maintain high population densities of Tylenchulus semipenetrans in field soil were carried out in microplots at two locations, and in an naturally infested orchard. Selections of Cleopatra mandarin (03) × Poncirus trifoliata (01) 03.01.5 and 03.01.13, Citrus volkameriana (23) × P. trifoliata 23.01.17, Troyer citrange (02) × Cleopatra mandarin 02.03.24, Troyer citrange × Common mandarin (04) 02.04.18, King mandarin (05) × P. trifoliata 05.01.7, and Carrizo citrange were exposed to continuous high population densities of a population of the Mediterranean biotype of T. semipenetrans. The selection 23.01.17 retained its resistance in the microplots and in the field (< 1.2% females and eggs per gram fibrous root of those on Carrizo citrange). The selection 03.01.5 also retained its resistance in the microplots at Moncada (< 0.5% females and eggs per gram fibrous root of those on Carrizo citrange) but numbers of females and eggs per gram fibrous root were 27% and 22% at Amposta, and 139% and 18% in the orchard of those on Carrizo citrange, respectively. The selection 05.01.7 supported equal number of females and 43% eggs per gram fibrous root of those on Carrizo citrange in the nematode-infested orchard. The remaining selections supported high populations of T. semipenetrans.

Population Densities of Tylenchulus semipenetrans Related to Physicochemical Properties of Soil and Yield of Clementine Mandarin in Spain

A survey was conducted from April to June 2002 in 62 orchards of clementine mandarin grafted on Troyer or Carrizo citrange rootstock in Catalonia (northeastern Spain) to determine the relationship between physicochemical soil properties and Tylenchulus semipenetrans population densities. Soil was analyzed to determine texture, pH, electric conductivity, organic matter content, N, P, K, Mg, calcium carbonate, and calcium oxide. Also, trials were conducted in 2003 and 2004 in three drip-irrigated orchards of clementine mandarin cv. Clemenules (El Pla and Mariclaire) or cv. Hernandina (Martorella) to determine the relationship between citrus nematode densities and yield of mandarin. T. semipenetrans was detected in 77% of the surveyed orchards. The number of second-stage juveniles + males in soil was related to N and K soil content (3.57953 - 0.001305 N + 0.00113 K, R2 = 0.1620, P < 0.0157). The relationship between relative yield of clementine mandarin cv. Clemenules and densities of females per gram of root in spring was described by the Seinhorst damage function model (0.035 + 0.965 (0.9995)(Pi - 287), R2 = 0.4782, P < 0.0001).

Microbiomes associated with infective stages of root-knot and lesion nematodes in soil.

Endoparasitic root-knot (Meloidogyne spp.) and lesion (Pratylenchus spp.) nematodes cause considerable damage in agriculture. Before they invade roots to complete their life cycle, soil microbes can attach to their cuticle or surface coat and antagonize the nematode directly or by induction of host plant defenses. We investigated whether the nematode-associated microbiome in soil differs between infective stages of Meloidogyne incognita and Pratylenchus penetrans, and whether it is affected by variation in the composition of microbial communities among soils. Nematodes were incubated in suspensions of five organically and two integrated horticultural production soils, recovered by sieving and analyzed for attached bacteria and fungi after washing off loosely adhering microbes. Significant effects of the soil type and nematode species on nematode-associated fungi and bacteria were revealed as analyzed by community profiling using denaturing gradient gel electrophoresis. Attached microbes represented a small specific subset of the soil microbiome. Two organic soils had very similar bacterial and fungal community profiles, but one of them was strongly suppressive towards root-knot nematodes. They were selected for deep amplicon sequencing of bacterial 16S rRNA genes and fungal ITS. Significant differences among the microbiomes associated with the two species in both soils suggested specific surface epitopes. Among the 28 detected bacterial classes, Betaproteobacteria, Bacilli and Actinobacteria were the most abundant. The most frequently detected fungal genera were Malassezia, Aspergillus and Cladosporium. Attached microbiomes did not statistically differ between these two soils. However, Malassezia globosa and four fungal species of the family Plectosphaerellaceae, and the bacterium Neorhizobium galegae were strongly enriched on M. incognita in the suppressive soil. In conclusion, the highly specific attachment of microbes to infective stages of phytonematodes in soil suggested an ecological role of this association and might be involved in soil suppressiveness towards them.

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.

Fungi recovered from root-knot nematodes infecting vegetables under protected cultivation

Abstract A survey was conducted in root-knot nematode-infested plastic houses to determine the diversity and frequency of occurrence of fungi associated with the nematode. The relationships between percentage fungal parasitism and physicochemical properties of soil were also investigated. Fifty-nine plastic houses were sampled in southeastern Spain, 42 treated with nematicides and 17 left untreated. Eleven fungal genera and unidentified fungi were isolated from nematode eggs or juveniles. Fungal parasitism occurred more frequently in untreated (82.4%) than treated (50%) soils. The species richness in untreated soils ranged from 0 to 5, the Shannon–Wiener diversity index (a measurement of how many different fungi there are in site taking into account how evenly they are distributed among the site) from 0 to 2.01, and the evenness index from 0.46 to 0.99. In treated soils, species richness ranged from 0 to 4, the Shannon–Wiener diversity index from 0 to 1.61, and the evenness index from 0.81 to 1. Of the sites with nematophagous fungi, Arthrobotrys dactyloides (34%), Cylindrocarpon sp., Neosartoria hiratsukae (17%), and Fusarium solani (14%) were the fungi most frequently found. Physicochemical properties of soil were similar in nematicide treated and untreated soils. Percent fungal parasitism in untreated soils correlated positively with lime, silt and carbonate content of soil.