Deciphering the Biology of \u3ci\u3eCryptophyllachora eurasiatica\u3c/i\u3e gen. et sp. nov., an Often Cryptic Pathogen of an Allergenic Weed, \u3ci\u3eAmbrosia artemisiifolia\u3c/i\u3e

A little known, unculturable ascomycete, referred to as Phyllachora ambrosiae, can destroy the inflorescences of Ambrosia artemisiifolia, an invasive agricultural weed and producer of highly allergenic pollen. The fungus often remains undetectable in ragweed populations. This work was conducted to understand its origin and pathogenesis, a prerequisite to consider its potential as a biocontrol agent. The methods used included light and transmission electron microscopy, nrDNA sequencing, phylogenetic analyses, artificial inoculations, and the examination of old herbarium and recent field specimens from Hungary, Korea, Ukraine and USA. The Eurasian and the North American specimens of this fungus were to represent two distinct, although closely related lineages that were only distantly related to other lineages within the Ascomycota. Consequently, we describe a new genus that includes Cryptophyllachora eurasiatica gen. et sp. nov. and C. ambrosiae comb. nov., respectively. The pathogenesis of C. eurasiaticawas shown in A. artemisiifolia. No evidence was found for either seed-borne transmission or systemic infection. Two hypotheses were developed to explain the interaction between C. eurasiatica and A. artemisiifolia: (i) as yet undetected seed-borne transmissions and latent, systemic infections; or (ii) alternative hosts

Deciphering the biology of Cryptophyllachora eurasiatica gen. et sp. nov., an often cryptic pathogen of an allergenic weed, Ambrosia artemisiifolia

A little known, unculturable ascomycete, referred to as Phyllachora ambrosiae, can destroy the inflorescences of Ambrosia artemisiifolia, an invasive agricultural weed and producer of highly allergenic pollen. The fungus often remains undetectable in ragweed populations. This work was conducted to understand its origin and pathogenesis, a prerequisite to consider its potential as a biocontrol agent. The methods used included light and transmission electron microscopy, nrDNA sequencing, phylogenetic analyses, artificial inoculations, and the examination of old herbarium and recent field specimens from Hungary, Korea, Ukraine and USA. The Eurasian and the North American specimens of this fungus were to represent two distinct, although closely related lineages that were only distantly related to other lineages within the Ascomycota. Consequently, we describe a new genus that includes Cryptophyllachora eurasiatica gen. et sp. nov. and C. ambrosiae comb. nov., respectively. The pathogenesis of C. eurasiatica was shown in A. artemisiifolia. No evidence was found for either seed-borne transmission or systemic infection. Two hypotheses were developed to explain the interaction between C. eurasiatica and A. artemisiifolia: (i) as yet undetected seed-borne transmissions and latent, systemic infections; or (ii) alternative hosts

A highly-simplified and inexpensive MALDI-TOF mass spectrometry sample-preparation method with broad applicability to microorganisms, plants, and insects

Matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry prepares proteins intact in the gas phase with predominantly a single positive charge. The times-of-flight of charged proteins along a tube held at high vacuum after acceleration in an electrical field are proportional to the square root of the mass-over-charge ratios for the proteins, thereby allowing a mass spectrum to be generated, which can then be used to characterize or identify a protein-containing sample. Several sample-preparation methods are currently available but not all of these are applicable to some forms of fungal biomass and few of these are well suited to the analysis of plant or insect material. We have therefore developed a simplified method that: lyses cells, selectively solubilizes basic proteins, dissolves matrix to a suitable concentration, generates spectra with good intensity and peak richness, costs no more (and generally less) than current methods, and is not constrained in terms of throughput by the availability of centrifuges. Using this method, and a reagent formulation comprising α-cyano-4-hydroxycinnamic acid matrix close to saturation in 60%–65% (v/v) acetonitrile in water containing 2.5% (v/v) trifluoroacetic acid, we have been able to differentiate between strains for a representative subset of aflatoxin-producing and aflatoxin-non-producing strains of Aspergillus fungi, to differentiate between Indian and Pakistani strains of Himalayan balsam rust, to differentiate between closely-related Crassula spp. and regional biotypes of Crassula helmsii, and to differentiate between rubbervine introduced into Australia and Brazil. We have also analyzed fall armyworm and stem-borer samples stored in 70% (v/v) ethanol and old dried insect specimens.

Molecular identification and pathogenicity assessment of a rust fungus infecting common ragweed (Ambrosia artemisiifolia) in its native North American range

A rust fungus collected from common ragweed (Ambrosia artemisiifolia) in Texas, USA, was identified as belonging to the Puccinia xanthii morphospecies based on its nrDNA ITS sequence. Pathogenicity studies carried out with this rust accession under quarantine conditions in the UK showed that the fungus was highly virulent on A. artemisiifolia plants from Australia. Recently, P. xanthii has been proposed as a potential classical biological control agent (CBCA) for common ragweed in its invasive range, focusing on Europe, despite previous doubts about its biocontrol potential. The results of the pathogenicity tests reported here support the suitability of this pathogen as a CBCA for common ragweed

Puccinia komarovii var. glanduliferae var. nov.: a fungal agent for the biological control of Himalayan balsam (Impatiens glandulifera)

Clearly defining the taxonomic identity of a potential biocontrol agent is an essential component of any biological control programme. As part of such a programme against Impatiens glandulifera, a highly invasive annual weed in both Europe and North America, the rust fungus Puccinia komarovii was collected on this host plant in its native Himalayan range. However, current literature indicates that P. komarovii is a pathogen of a number of Impatiens species globally and was described originally from I. parviflora, a species native to Asia and now naturalized in Europe. Morphological comparisons, based on urediniospore and teliospore measurements, were generally inconclusive in showing any clear differences between the accession from I. glandulifera and those from other Impatiens species. Both, nrDNA ITS and ITS2-LSU sequence analyses indicated a difference between the rust infecting I. glandulifera compared to accessions on other hosts. However, the large variations in both ITS and 28S (ITS2-LSU) sequences determined within single accessions in this study, makes a clear separation difficult. Cross-inoculation experiments, using one accession of P. komarovii ex I. glandulifera (from India) and two accessions of P. komarovii ex I. parviflora (from China and Hungary), confirmed the specificity of these strains to their original hosts. Two Himalayan Impatiens species, I. scabrida and I. brachycentra, showed varying levels of susceptibility to these rust accessions, where the former was weakly susceptible to all three accessions and the latter was weakly susceptible only to P. komarovii ex I. parviflora (from China). However, commercial cultivars of I. balsamina proved to be fully susceptible to all rust accessions, although this has not been demonstrated under field conditions in India. Based on these host specificity differences between the rust accessions, we propose a new variety: Puccinia komarovii var. glanduliferae var. nov. associated with I. glandulifera in the Himalayas