Rosa H. Manzanilla-López

The Pochonia chlamydosporia Serine Protease Gene vcp1 Is Subject to Regulation by Carbon, Nitrogen and pH: Implications for Nematode Biocontrol

The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances.

Measuring abundance, diversity and parasitic ability in two populations of the nematophagous fungus Pochonia chlamydosporia var. chlamydosporia

Abstract Abundance, genetic diversity and parasitic ability in the facultative nematode parasite Pochonia chlamydosporia var. chlamydosporia were compared in soils from two sites in Portugal under long-term tomato cultivation where root-knot nematodes (Meloidogyne sp.) were present. Fungal abundance assessed by selective agar or real-time quantitative PCR with specific primers was similar in both soils. PCR fingerprinting of isolates with ERIC primers indicated that the dominant P. c. var. chlamydosporia biotypes (profiles A and B) in both soils were very closely related, although a second biotype (profile C) was detected in one soil. When tomato plants infected with M. incognita were grown in the two soils, only profiles A and B were recovered from eggs. Primers based on polymorphisms in vcp1 demonstrated that isolates with profiles A and B were likely to prefer root-knot nematodes, whereas profile C preferred cyst nematodes. In the soil containing profiles A, B and C, egg parasitism by P. chlamydosporia was estimated at 1% using water agar plates with antibiotics but fewer than 0.2% of M. incognita eggs were shown to be infected with P. c. var. chlamydosporia when using species-specific β-tubulin-PCR primers. In contrast, the soil containing only profile B showed 22% egg parasitism on water agar plates and more than 2.5% of eggs were confirmed as P. c. var. chlamydosporia by species-specific β-tubulin-PCR primers. The results, which reveal limited diversity within the fungus at the two sites, are discussed in relation to biological control of plant-parasitic nematodes.

A molecular diagnostic method for detecting Nacobbus in soil and in potato tubers

Species of the genus Nacobbus have the potential to reduce yields of major food crops such as potato, sugar beet and tomato in many parts of the world, thus warranting a quarantine effort to avoid their introduction. Molecular studies offer a new method for routine quarantine diagnostics for this nematode that will be faster and more sensitive than previous methods. A primer set was designed from Nacobbus ITS sequences and their specificity confirmed. DNA was extracted from nematodes, soil and potato tubers for use in PCR. Optimised PCR conditions were established and the PCR products were separated on 2% agarose gels, showing that specific ITS primers for the detection of Nacobbus generated a single PCR product, although band size varied slightly between species and soil isolates. The product was generated from DNA extracted from all the Nacobbus samples but not from other nematodes tested (Pratylenchus, Radopholus, Meloidogyne, Globodera, Heterodera). No bands were generated from the uninfested control soil and control tuber DNA samples, thus demonstrating the specificity of the primers. For the first time, Nacobbus was detected in soil and tuber samples using molecular approaches. These results have important applications not only in analysing advisory samples but also in the screening of material for quarantine purposes.

Rapid and reliable DNA extraction and PCR fingerprinting methods to discriminate multiple biotypes of the nematophagous fungus Pochonia chlamydosporia isolated from plant rhizospheres

Aims:  To develop a simple, rapid, reliable protocol producing consistent polymerase chain reaction (PCR) fingerprints of Pochonia chlamydosporia var. chlamydosporia biotypes for analysing different fungal isolates during co‐infection of plants and nematodes.

Re-evaluation of the life-cycle of the nematode-parasitic bacterium Pasteuria penetrans in root-knot nematodes, Meloidogyne spp.

Comparisons of the growth of Pasteuria penetrans in adult root-knot nematode females infected with P. penetrans dissected from the roots of tomato plants were undertaken using bright-field and scanning electron microscopy. Samples of infected females were nutritionally compromised by maintaining them in sterile saline at 30°C for different periods of time following their removal from the root system. Observations of these females maintained in saline revealed a series of growth stages of Pasteuria hitherto not documented, consisting of rhizoids, rod-like bacilli and granular masses. A new life-cycle for Pasteuria is described consisting of three phases: Phase I: attachment and germination; Phase II: rhizoid production and exponential growth; and Phase III: sporogenesis. These newly observed stages of the life cycle show a high degree of similarity to the developmental stages seen in other Bacillus spp.

Nacobbus aberrans (Thorne, 1935) Thorne & Allen, 1944 (Nematoda: Pratylenchidae); a nascent species complex revealed by RFLP analysis and sequencing of the ITS-rDNA region

Twelve populations of Nacobbus aberrans, sensu lato, from Mexico, Bolivia, Peru, Ecuador and Argentina were subjected to molecular analysis of their genetic variability. RFLP banding patterns revealed three groups: i) Mexico, Ecuador and Argentina 1 populations; ii) Bolivia and Peru populations; iii) Argentina 2 population. These differences were confirmed by sequencing the ITS rDNA region. Depth of branching was strongly supportive of the presence of three separate species, thus supporting the hypothesis that N. aberrans s.l. is indeed a species complex. The populations from Mexico, Ecuador and Argentina 1 are attributed to N. aberrans s.s., although this requires confirmation by molecular characterisation of N. aberrans from the type locality in the USA; those from Bolivia and Peru are attributed to N. bolivianus Lordello, Zamith & Boock, 1961 with Argentina 2 regarded as representing another taxon. Nacobbus serendipiticus and N. batatiformis are removed from synonymy under N. aberrans s.l . and regarded as species inquirendae . Consistent minor banding patterns in the RFLP profiles may indicate that the genus reproduces predominantly by parthenogenesis.

Identification and molecular characterisation of new Mexican isolates of Pochonia chlamydosporia for the management of Meloidogyne spp.

New Mexican isolates of the nematophagous fungus Pochonia chlamydosporia were obtained from nematode infested fields in the vegetable growing area of Tepeaca Valley, Puebla State, Mexico. Based on macro and microscopic morphology, seven ‘putative’ P. chlamydosporia isolates were selected and the DNA extracted for polymerase chain reaction (PCR). Three new isolates of P. chlamydosporia were identified: Pcp2, Pcp21 and Pcp31. The amplification reaction of the internal transcribed spacer (ITS) region revealed a 650 bp amplicon which was used in a maximum likelihood phylogenetic inference analysis. Three groups were recovered in the tree topology, supported by a > 90% bootstrap value. Nucleotide identity values were > 83.6% between the test sequences and the reference sequence. In addition, using specific primers for two existing varieties of P. chlamydosporia, restriction fragment length polymorphism on the ITS products in conjunction with the phylogenetic inferences and the molecular test for detection of P. chlamydosporia vcp1 gene, it was found that all three isolates belong to a new variety which we have named P. chlamydosporia var. mexicana. We compared the chlamydospore production rate, rhizosphere colonisation and egg parasitism percentages of the three native isolates in Meloidogyne spp. with a reference isolate (Pc10). Native isolates produced > 1×106 chlamydospores/50 g of substrate (of which more than 80% were viable), colonised > 80% of the rhizosphere, and parasitised > 60% of Meloidogyne incognita and Meloidogyne arenaria eggs. Meloidogyne hapla egg parasitism was < 60%. Isolates Pcp2 and Pcp21 were identified as potential biological control agents of Meloidogyne spp. to be tested further in greenhouse and field tests.

Speciation within Nacobbus: consilience or controversy?

Although Nacobbus aberrans , the false root-knot nematode, is endemic to the American continent, it justifies the attention granted by international quarantine regulations due to its broad host-range and economic importance in crops such as potato, tomato and sugarbeet. Until recently, it was widely accepted that the genus Nacobbus included only two valid species: N. aberrans and N. dorsalis . Traditionally, criteria to identify and separate the species have been based on morphology, host-range and some biological features. Molecular characterisation of populations can help to identify species that are otherwise almost impossible to segregate and has provided information that has identified aggregates of species or groups within the N. aberrans complex. This information needs to be integrated with knowledge already provided by non-molecular methodologies in order to establish a robust and workable framework that facilitates the assessment and assignment of nematode populations to an identifiable species, subspecies or group. The current molecular data indicate that the traditional two species paradigm is outdated and demand a re-assessment of the taxonomic status of the N. aberrans species complex. Whether molecular-based clades are recognised at specific rank or not, sequence analysis opens the door to reliable and incontrovertible diagnostics, a prerequisite for sustainable management practices and international quarantine regulations pertaining to the genus.

Epidemiology and integrated control of Nacobbus aberrans on tomato in Mexico

Population densities, population fluctuations, yield loss and disease incidence caused by Nacobbus aberrans on tomato (Lycopersicon esculentum) were studied, using an epidemiological approach, in a field experiment that included three different control regimes: an integrated control (IC) scheme, which included fertilisation, nematicide (ethoprop) and chicken manure; a technical control (TC) scheme, based on the best local practices of fertilisation and use of carbofuran for nematode control; and a check treatment (AC), with no application of fertilisers or nematicide. At least three generations of N. aberrans occurred through the cropping season and the numbers of galls/plant and females/g of root through the crop season were used to define the area under a disease progress curve (AUDPC). The variables b−1 (Weibulls apparent infection rate), AUDPC and Yf (final disease incidence) indicated less crop damage under the IC scheme than under the other two schemes (TC and AC). The IC scheme resulted in increased plant height (41-49%), foliage dry weight (37-53%) and stem diameter (31-41%) compared with the TC and AC schemes. Tomato yields in IC surpassed those from TC and AC by 34 and 83%, respectively, while TC exceeded AC by 73%. The yield loss attributed to N. aberrans was 12, 29 and 83% in IC, TC and AC, respectively. The IC scheme improved commercial production by 20 and 81% in comparison to the TC and AC schemes. This was largely due to effective control of the initial inoculum density, which affects the first generation of the nematode population; control of this generation is essential for avoidance of yield loss. The first generation is completed during the period 0-60 dat (days after transplanting), i.e., during the critical stages of flowering, fruit initiation and fruit set (40, 50 and 60 dat). Data on plant performance taken every 10 days were used to derive a multiple point model for calculation of production loss.

A combined cryo-scanning electron microscopy/cryoplaning approach to study the infection of Meloidogyne incognita eggs by Pochonia chlamydosporia

The fungus Pochonia chlamydosporia is a saprophytic soil-dwelling fungus and is also a parasite of the eggs of the root-knot plant-parasitic nematodes (Meloidogyne spp.). Studies on its molecular characterisation, diversity, tritrophic interactions and ecology have been carried out. However, to elucidate the role in soil and rhizosphere ecology of this and other nematophagous fungi used in biological control, and to enhance their exploitation, it is necessary to improve the understanding of the biology and fungus-nematode infection process using different approaches, including microscopy. Low-temperature Scanning Electron Microscopy (cryo-SEM) techniques allow the examination of frozen, fully hydrated samples that can reveal important ultrastructural features occurring through fungus-nematode interactions. A method that combined cryo-SEM with cryoplaning was developed to examine samples of eggs, gelatinous matrix and females of Meloidogyne incognita colonised by P. chlamydosporia. The fungal samples were produced in potato dextrose agar to which different nematode stages were added and processed for cryo-SEM and cryoplaning within a period of 0-72 h post inoculation. The method was found to be rapid and economical, provided clear and detailed external images of the infection process and allowed viewing of sections through structures with minimal processing in comparison to other cryo-SEM techniques and it could be adapted to study other fungus-nematode interactions.