Lectera, a new genus of the Plectosphaerellaceae for the legume pathogen Volutella colletotrichoides

Volutella colletotrichoides is shown to belong to the Plectosphaerellaceae rather than the Hypocreales where other species of that genus reside. The new genus Lectera is described for V. colletotrichoides, and for a further, previously undescribed species with slightly longer conidia and differences in rDNA ITS and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences. Lectera species are found to be soil-dwelling organisms associated particularly with species of Fabaceae, and L. colletotrichoides is demonstrated to be a pathogen of Cicer arietinum and other grain legumes.


Introduction
Volutella colletotrichoides was first described from diseased alfalfa and other forage legumes collected from Iowa, USA (Chilton 1954), evident as pink acervular dark-setose fruit bodies erumpent from stems and leaves.It was compared with four other species of Volutella known to be associated with legumes, and distinguished by its acervular setae that are dark rather than hyaline, and by the size of its conidia.The dark setae and relatively large conidia invited comparison with Colletotrichum and led to choice of the specific epithet.A collection with especially numerous setae was given varietal status as V. colletotrichoides var.setosa.The pathogenicity of Volutella colletotrichoides was demonstrated by observations of naturally infected plants in the glasshouse and in the field, and also by infection experiments (Chilton 1954).The original samples derived from stem cuttings of Medicago sativa (alfalfa), Trifolium pratense (red clover) and Lotus corniculatus (bird's foot trefoil), with Medicago plants proving the most vulnerable to infection, and were reproduced in plant samples transferred to Lincoln, Nebraska.Periodic observations over the following two years showed sparse and erratic occurrence of the pathogen, suggesting that the fungus is not a virulent pathogen, at least in those local conditions.Chilton found that inoculations on mature alfalfa and red clover plants produced petiole girdling and girdling of the stem tips, causing a blighted appearance in severely infected plants.Infection of mature stem tissues produced elliptical sunken lesions to a limited extent.No evidence of systemic invasion of the plant was found although stem infections gradually enlarged to involve much of the plant under high moisture conditions.
Host range experiments (Chilton 1954) provided evidence that Trifolium hybridum and Medicago falcata were susceptible to infection, in addition to the three plant species on which the original observations were made.In contrast, Melilotus alba and Glycine max were not infected under similar experimental conditions.As the plants originally infected were of foreign origin (Chilton did not specify from where) and the occurrence of the pathogen was highly localized, it seems likely that Volutella colletotrichoides was a pathogen introduced into the USA that did not become established, rather than a native species.There are no further records of the species from the USA, and no material in the national fungus collection BPI (Farr and Rossman 2011).
Published reports of Volutella colletotrichoides from other regions are sparse.It was reported from Glycine max in Ethiopia by Mengistu and Sinclair (1979), from the same host (as G. ussuriensis) in Democratic Republic of the Congo by Lenné (1990), and on Senna sophera from India (Lenné 1990).Eken et al. (2002) showed the fungus to be present on alfalfa in Turkey.A sequence from an unlocalised sample on Viola sp. was submitted to GenBank (http://www.ncbi.nlm.nih.gov/nuccore/AJ301962.1)by Hagedorn (unpubl., 2002), and cultures from alfalfa from Turkey (reported above) and South Africa are stored in the CBS culture collection (http://www.cbs.knaw.nl/).The plant associates given above do not necessarily imply a biological relationship.
In the winter of 1988, a field survey was carried out for diseases of chickpea (Cicer arietinum) in the vicinity of Salheia, at that time a newly reclaimed region between the Nile Delta and the Suez Canal (Ismaelia) in northern Egypt.There, blighted chickpea plants were observed and collected, with the causal organism initially assumed to be the common chickpea pathogen Ascochyta rabiei.The macroscopic visible symptoms were circular or elongated dark brown to black anthracnose-like lesions approximately one centimeter long on the lower stem parts in addition to partly chlorotic leaves.The causal organism was isolated and sent to the Danish Government Institute of Seed Pathology for Developing Countries (DGISP), and designated as DGISP 271.Later the diagnostic studies were transferred to The Danish Veterinary and Agricultural University.Here the organism was registered as CP 2035.After an extended period of analysis using both morphological and molecular methods with multiple collaborators, the affinities of CP 2035 have finally been confirmed to be with V. colletotrichoides.
As Chilton (1954) had observed, we found that Volutella colletotrichoides differed in a number of morphological characteristics from other Volutella species.As molecular analysis has confirmed this separation, we therefore describe the new genus Lectera for V. colletotrichoides and a similar, closely related species.

Materials and methods
Isolates were retrieved from storage in the CABI Genetic Resources Collection (see Table 1).For morphological analysis, strains were grown at 25°C for 7 d on PCA and PDA media (Smith and Onions 1994).Observations were made using microscope preparations in water and lactic acid, with measurements made from slides mounted in lactic acid.
Infection studies were carried out, using chickpea cultivar Family 88 as well as certain other species of Fabaceae as hosts: Vicia faba, Glycine max, Pisum sativum, Vigna unguiculata, and Phaseolus vulgaris.The inoculum was produced as above.Stems and leaves were inoculated without prior wounding using a spore suspension produced by flooding four week old cultures with 10 ml sterile distilled water.The conidial concentration was adjusted to 2 × 10 5 ml -1 and finally one drop of Tween 20 (as detergent) was added.Symptoms were observed two weeks after inoculation.
For the molecular analysis, strains were grown on malt extract (MADW) agar plates (Smith and Onions 1994) at 28°C for 5-7 days.The isolates were subcultured onto fresh MADW plates and incubated at 28°C for 7-10 days prior to DNA extraction.
Aliquots (4 µl) of amplification products were assessed for quality by gel electrophoresis using 1.5 % Seakem LE agarose (BMA, UK) for 2 h at 5V cm -1 in half- Remaining unused PCR products were purified with the microCLEAN PCR Purification Kit (Microzone Ltd, UK) following the manufacturer's instructions.The purified PCR products were utilised in sequencing reactions undertaken in a Primus 96 plus thermal cycler (MWG-BIOTECH AG, Germany) by using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) with primer TW81 (as above).Sequencing conditions were: 96°C for 1 min followed by 25 cycles of 20 s at 96°C, 10 s at 50°C, 4 min at 60°C (ramp rate: 1°C s -1 ).Excess unincorporated BigDye was removed with DyeEx 2.0 affinity columns (Qiagen Ltd., UK) according to the manufacturer's instructions and the sequencing reaction products were suspended in HiDi Formamide (Applied Biosystems, UK).These products were separated on a capillary array 3130 Genetic Analyser (Applied Biosystems, UK).
Sequence trace files were first assessed for quality using Sequencing Analysis Software v5.2 Patch 2 (Applied Biosystems, UK) and then checked manually using the software package Chromas v2.23 (Technelysium, Australia) and exported as text files.Sequences were deposited in GenBank and Accession numbers obtained (see Table 1).
Sequences of studied species were aligned using the default parameters of Clustal W (Thompson et al. 1994), then optimised manually using the CLUSTALW plug-in of MEGA5 (Tamura et al. 2011).Phylogenetic inferences were made from Neighbour Joining trees constructed from Maximum Likelihood in Mega5.Branch support was estimated by bootstrap analysis (1000 replicates).Accession numbers of rDNA ITS and GAPDH sequences derived from the public databases are cited on the phylogenetic trees.

Pathogenicity
Besides Cicer arietinum (from which the tester strain CP2035 (IMI 332702) was isolated), the following leguminous plants were found to function as hosts after experimental inoculation: Glycine max, Phaseolus vulgaris, Pisum sativum, Vicia faba and Vigna unguiculata.The main symptoms were evident as brown to black lesions on the lower stem parts, within which acervuli develop.Leaves also showed similar symptoms in all species, in addition to chlorosis.The symptoms were very similar to those caused by Ascochyta rabiei, a well-known and commonly occurring pathogen causing ascochyta blight on chickpea.taxonomy Lectera P.F.Cannon, gen.nov.http://species-id.net/wiki/LecteraMycobank: MB 550041 Etymology.Named after Dr. Hannibal Lecter (Harris 1988), another aggressive organism with a liking for fava beans (Vicia faba).
Conidiomata intermediate between sporodochia and acervuli, erumpent through host tissuesand without a clear upper wall, hemispherical to ± globose, pink or fleshcoloured, accompanied by dark brown septate tapering, primarily marginal, setae.Conidia formed from hardly modified conidiogenous cells, hyaline, aseptate, slightly curved, smooth, fusiform with pointed ends.Appressoria formed after conidial germination, dark brown, round to ovate with smooth margins.
Description.Conidiomata formed at the apex of a short peg-like vegetative structure that is erumpent through host tissues, 80-350 µm diam, cushion-shaped to almost globose, with a compact palisade of conidiogenous cells usually surrounded by setae.
Interactions.Strains of Lectera colletotrichoides have been demonstrated to be pathogenic towards a range of Fabaceae species, but they are also commonly found associated with plants from other families and isolated from soil and plant litter.It also grows well in standard agar culture.It therefore can be presumed to exist (and probably grow actively) as a saprobe, and it is possible that the non-legume isolates originate from soils used to grow legumes in rotation.
Conservation assessment.The species as currently circumscribed has not been reported since 2002 but is very widely distributed, is associated with a wide range of plant taxa and apparently can exist as a saprobe in soil and leaf litter without a direct plant association.It does not appear to be an economically important pathogen except perhaps in limited circumstances.However, as there is a high risk of confusing disease symptoms of Lectera colletotrichoides with those caused by Ascochyta rabiei, the economic impact of Lectera colletotrichoides may be underestimated.It may be sensitive to agricultural pesticides, but is unlikely to face major threats from specific eradication measures.Its conservation status (Dahlberg and Mueller 2011)  Description.Differs from Lectera colletotrichoides by its longer conidia (7.8-10 × 2-2.5 µm; mean 8.87 µm, sd 0.56, n=20) with a mean length/breadth ratio of 3.98: 1, with three short insertions and a single substitution in the ITS sequence.
Host species.Associated with Triticum sp.(Poaceae).Distribution.Only definitely known from the type locality.
Interactions.No details are known; the data associated with the type do not indicate whether the fungus was thought to be pathogenic.
Conservation assessment.The species is only known from a single collection made in 1974, though a strain isolated from Viola sp. and identified as Volutella colletotrichoides with an identical ITS sequence (AJ301962) was deposited in the BBLF culture collection as BBA 71246.Its geographical origin is unknown, and the status of the living culture is uncertain.Its conservation status (Dahlberg and Mueller 2011) must be considered as Data Deficient, but is potentially Critically Endangered.

Discussion
The genus Volutella is poorly researched and no modern monograph is available, but some of the more well-known species, including the type V. ciliata, were included in a recent phylogenetic study of Fusarium-like fungi (Gräfenhan et al. 2011).This work demonstrated that Volutella (as represented by its type) occupies a distinct clade within the Nectriaceae, with Chaetopsina as sister group and Pseudonectria buxi (the anamorph of which was at one time ascribed to Volutella) also related.Volutella in its currently understood sense is clearly polyphyletic (Seifert et al. 2011), with many of its constituent species referable to other genera.
Lectera has brightly coloured sporodochia surrounded by brown setae (those in true Volutella species are hyaline).Other sporodochial genera with these characteristics include Kutilakesa, Sarcopodium and Actinostilbe (Seifert et al. 2011).The taxonomic affinities of the type of Kutilakesa are currently unclear (no sequences are available), but the only other species in that genus has a Nectriella teleomorph (Alfieri and Samuels 1979) and is therefore likely to belong to the Hypocreales.The type of Sarcopodium belongs to the Lanatonectria clade (Nectriaceae, Hypocreales) according to Summerbell et al. (2011).Both Kutilakesa and Sarcopodium have flexuous verruculose setae with rounded apices, in contrast to Lectera, where they are straight, hardly ornamented and tapering to an acute tip.On one occasion, a teleomorphic fungus with thin-walled asci and aseptate ascospores was observed on old wilted plant material infected by L. colletotrichoides, but the observation could not be repeated and we are not confident that the two fungi are genetically connected.
One other species of Volutella has orange sporodochia surrounded by brown tapering setae, in common with Lectera colletotrichoides, V. melaloma Berk.& Br. which was described from dead leaves of Carex in the UK (Berkeley and Broome 1850).It is possible that this species is congeneric with L. colletotrichoides, but it differs in a number of important characters: the conidiogenous cells are thicker-walled and do not taper, the setae are longer, more closely septate and smooth-walled, and the conidia are significantly larger and more tapered, measuring 15-18 × 3.5-4.5 µm.No recently collected material is available for sequencing.
The ITS sequences generated in this study strongly suggest that Lectera has affinities with the Plectosphaerellaceae, and may be a sister group to Verticillium (Fig. 1), although this relationship only receives weak bootstrap support.That systematic relationship was previously noted incidentally by Réblová and Seifert (2004), while researching into the phylogeny of Conioscypha and its relatives.Many fungi in these groups are wide-spectrum soil-borne plant pathogens, in common with Lectera.The Plectosphaerellaceae includes a series of Acremonium-like fungi as well as Plectosphaerella, Verticillium, Gibellulopsis and Musicillium (Zare et al. 2004(Zare et al. , 2007)).Verticillium has recently been monographed using multigene phylogenetic data (Inderbitzin et al. 2011), but its generic position was not assessed.Based on LSU and SSU rDNA data, Réblová et al. (2011) considered the Plectosphaerellaceae to be well-defined, and a sister group with the Glomerellales to the Microascales, an order containing important plant pathogens such as Ceratocystis.
Lectera colletotrichoides contains two distinct ITS phylotypes, IMI 265740 differing from the other strains sequenced by only a single base pair.The ITS sequence of L. longa differs from L. colletotrichoides by three short insertions and a single substitution, and this combined with differences in conidial length and lengh/width ratio justifies its separation.The distinction between the two species is supported by GAPDH sequences also (see fig. 3).The gene fragment sequenced here is a ~200 bp intron in the glyceraldehyde 3-phosphate dehydrogenase gene, used for phylogenetic studies of  Colletotrichum by Damm et al. (2009) and Guerber et al. (2003), not the same fragment used for Verticillium by Inderbitzin et al. (2011).
The wide distribution of Lectera colletotrichoides and its association with agricultural ecosystems make it difficult to assess its geographical origin, although its apparent preference for dry-land legumes might indicate an evolutionary history centred on the Near East.However, judging from environmental sequencing studies, it (or a closely related species) does seem to be present in soils in SE USA (Wu et al. 1997, Jackson 2010), suggesting that its distribution in that country is not as restricted as indicated by Chilton (1954).

Figure 1 .
Figure 1.ML ITS phyogram showing the phylogenetic position of Lectera species.