This work was supported by a United Kingdom Science Research Council grant to N. J. Mackintosh and A. Dickinson. We would like to thank A. Dickinson for his advice and for comments on this manuscript and D. J. Nicholas for invaluable assistance in running the animals. We are also grateful to R. Hammans for producing the figures and to R. Hutchison and J. Read for their care of the animals.

No abstract

The host-specific plant pathogen Pseudomonas syringaeelicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity inA 230 assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringaepathovars, Pseudomonas viridiflava, Ralstonia(Pseudomonas) solanacearum, andXanthomonas campestris. ΔhrpZ::nptII orhrpW::ΩSpr P. syringaepv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in ahrpZ hrpW double mutant. These features of hrpWand its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.

The species Pseudomonas syringae encompasses plant pathogens with differing host specificities and corresponding pathovar designations. P. syringae requires the Hrp (type III protein secretion) system, encoded by a 25-kb cluster ofhrp and hrc genes, in order to elicit the hypersensitive response (HR) in nonhosts or to be pathogenic in hosts. DNA sequence analysis of the hrpC and hrpRSoperons of P. syringae pv. syringae 61 (brown spot of beans), P. syringae pv. glycinea U1 (bacterial blight of soybeans), and P. syringae pv. tomato DC3000 (bacterial speck of tomatos) revealed that the 13 genes comprising the right half of the hrp cluster (including those in the previously sequenced hrpZ operon) are conserved and identically arranged. The hrpC operon is comprised of hrpF,hrpG, hrcC, hrpT, and hrpV. hrcC encodes a putative outer membrane protein that is conserved in all type III secretion systems. The other four genes appear to be characteristic of group I Hrp systems, such as those possessed byP. syringae and Erwinia amylovora. The predicted products of these four genes in P. syringae pv. syringae 61 are HrpF (8 kDa), HrpG (15.4 kDa), HrpT (7.5 kDa), and HrpV (13.4 kDa). HrpT is a putative outer membrane lipoprotein. HrpF, HrpG, and HrpV are all hydrophilic proteins lacking N-terminal signal peptides. The HrpG, HrcC, HrpT, and HrpV proteins of P. syringae pathovars syringae and tomato (the two most divergent pathovars) had at least 76% amino acid identity with each other, whereas the HrpF proteins of these two pathovars had only 36% amino acid identity. The HrpF proteins of P. syringae pathovars syringae and glycinea also showed significant similarity to the HrpA pilin protein of P. syringae pathovar tomato. Functionally nonpolar mutations were introduced into each of the genes in thehrpC operon of P. syringae pv. syringae 61 by insertion of an nptII cartridge lacking a transcription terminator. The mutants were assayed for their ability to elicit the HR in nonhost tobacco leaves or to multiply and cause disease in host bean leaves. Mutations in hrpF, hrcC, andhrpT abolished or greatly reduced the ability of P. syringae pv. syringae 61 to elicit the HR in tobacco. ThehrpG mutant had only weakly reduced HR activity, and the activity of the hrpV mutant was indistinguishable from that of the wild type. Each of the mutations could be complemented, but surprisingly, the hrpV subclone caused a reduction in the HR elicitation ability of the ΔhrpV::nptIImutant. The hrpF and hrcC mutants caused no disease in beans, whereas the hrpG, hrpT, and hrpV mutants had reduced virulence. Similarly, thehrcC mutant grew little in beans, whereas the other mutants grew to intermediate levels in comparison with the wild type. These results indicate that HrpC and HrpF have essential functions in the Hrp system, that HrpG and HrpT contribute quantitatively but are not essential, and that HrpV is a candidate negative regulator of the Hrp system.

The casing material required in mushroom cultivation presents a very rich ecological niche, which is inhabited by a diverse population of bacteria and fungi. In this work three different casing materials, blonde peat, black peat and a 50 : 50 mixture of both, were compared for their capacity to show a natural suppressive response against dry bubble, Lecanicillium fungicola (Preuss) Zare and Gams, and wet bubble, Mycogone perniciosa (Magnus) Delacroix. The highest mushroom production was collected from crops cultivated using the mixed casing and black peat, which were not significantly different in yield. However, artificial infection with mycoparasites resulted in similar yield losses irrespective of the material used, indicating that the casing materials do not confer advantages in disease suppression. The composition of the microbiome of the 50 : 50 casing mixture along the crop cycle and the compost and basidiomes was evaluated through next-generation sequencing (NGS) of the V3-V4 region of the bacterial 16S rRNA gene and the fungal ITS2 region. Once colonized by Agaricus bisporus, the bacterial diversity of the casing microbiome increased and the fungal diversity drastically decreased. From then on, the composition of the casing microbiome remained relatively stable. Analysis of the composition of the bacterial microbiome in basidiomes indicated that it is highly influenced by the casing microbiota. Notably, L. fungicola was consistently detected in uninoculated control samples of compost and casing using NGS, even in asymptomatic crops. This suggests that the naturally established casing microbiota was able to help to suppress disease development when inoculum levels were low, but was not effective in suppressing high pressure from artificially introduced fungal inoculum. Determination of the composition of the casing microbiome paves the way for the development of synthetic casing communities that can be used to investigate the role of specific components of the casing microbiota in mushroom production and disease control.