Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression.

Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers' deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus's central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS's function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion.IMPORTANCEBrown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of "omics" platforms has recently uncovered the key "oxidative-hydrolytic" mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes' catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system's use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

Global field collection data confirm an affinity of brown rot fungi for coniferous habitats and substrates.

Unlike 'white rot' (WR) wood-decomposing fungi that remove lignin to access cellulosic sugars, 'brown rot' (BR) fungi selectively extract sugars and leave lignin behind. The relative frequency and distribution of these fungal types (decay modes) have not been thoroughly assessed at a global scale; thus, the fate of one-third of Earth's aboveground carbon, wood lignin, remains unclear. Using c. 1.5 million fungal sporocarp and c. 30 million tree records from publicly accessible databases, we mapped and compared decay mode and tree type (conifer vs angiosperm) distributions. Additionally, we mined fungal record metadata to assess substrate specificity per decay mode. The global average for BR fungi proportion (BR/(BR + WR records)) was 13% and geographic variation was positively correlated (R2 = 0.45) with conifer trees proportion (conifer/(conifer + angiosperm records)). Most BR species (61%) were conifer, rather than angiosperm (22%), specialists. The reverse was true for WR (conifer: 19%; angiosperm: 62%). Global BR proportion patterns were predicted with greater accuracy using the relative distributions of individual tree species (R2 = 0.82), rather than tree type. Fungal decay mode distributions can be explained by tree type and, more importantly, tree species distributions, which our data suggest is due to strong substrate specificities.

The genus Fomitopsis (Polyporales, Basidiomycota) reconsidered.

Based on seven- and three-gene datasets, we discuss four alternative approaches for a reclassification of Fomitopsidaceae (Polyporales, Basidiomycota). After taking into account morphological diversity in the family, we argue in favour of distinguishing three genera only, viz. Anthoporia, Antrodia and Fomitopsis. Fomitopsis becomes a large genus with 128 accepted species, containing almost all former Fomitopsis spp. and most species formerly placed in Antrodia, Daedalea and Laccocephalum. Genera Buglossoporus, Cartilosoma, Daedalea, Melanoporia, Neolentiporus, alongside twenty others, are treated as synonyms of Fomitopsis. This generic scheme allows for morphologically distinct genera in Fomitopsidaceae, unlike other schemes we considered. We provide arguments for retaining Fomitopsis and suppressing earlier (Daedalea, Caloporus) or simultaneously published generic names (Piptoporus) considered here as its synonyms. Taxonomy of nine species complexes in the genus is revised based on ITS, ITS + TEF1, ITS + TEF1 + RPB1 and ITS + TEF1 + RPB2 datasets. In total, 17 species are described as new to science, 26 older species are reinstated and 26 currently accepted species names are relegated to synonymy. A condensed identification key for all accepted species in the genus is provided. Taxonomic novelties: New species: Fomitopsis algumicola Grebenc & Spirin, F. caseosa Vlasák & Spirin, F. cupressicola Vlasák, J. Vlasák Jr. & Spirin, F. derelicta Vlasák & Spirin, F. dollingeri Vlasák & Spirin, F. fissa Vlasák & Spirin, F. lapidosa Miettinen & Spirin, F. lignicolor Vlasák & Spirin, F. maculosa Miettinen & Spirin, F. pannucea Runnel & Spirin, F. perhiemata Viner & Spirin, F. purpurea Spirin & Ryvarden, F. retorrida Spirin & Kotiranta, F. solaris Rivoire, A.M. Ainsworth & Vlasák, F. tristis Miettinen & Spirin, F. tunicata Miettinen & Spirin, F. visenda Miettinen & Spirin. New combinations: Fomitopsis aculeata (Cooke) Spirin & Miettinen, F. aethalodes (Mont.) Spirin, F. alaskana (D.V. Baxter) Spirin & Vlasák, F. albidoides (A. David & Dequatre) Bernicchia & Vlasák, F. amygdalina (Berk. & Ravenel) Spirin & Vlasák, F. angusta (Spirin & Vlasák) Spirin & Vlasák, F. atypa (Lév.) Spirin & Vlasák, F. caespitosa (Murrill) Spirin & Miettinen, F. calcitrosa (Spirin & Miettinen) Spirin & Miettinen, F. circularis (B.K. Cui & Hai J. Li) Spirin, F. concentrica (G. Cunn.) M.D. Barrett, F. cyclopis (Miettinen & Spirin) Miettinen & Spirin, F. dickinsii (Berk. ex Cooke) Spirin, F. elevata (Corner) Spirin & Miettinen, F. eucalypti (Kalchbr.) Spirin, F. ferrea (Cooke) Spirin & Viner, F. flavimontis (Vlasák & Spirin) Vlasák & Spirin, F. foedata (Berk.) Spirin & Miettinen, F. gilvidula (Bres.) Spirin & Miettinen, F. glabricystidia (Ipulet & Ryvarden) Miettinen & Ryvarden, F. globispora (Ryvarden & Aime) Spirin, F. hartmannii (Cooke) M.D. Barrett & Spirin, F. hyalina (Spirin, Miettinen & Kotir.) Spirin & Miettinen, F. hypoxantha (Bres.) Spirin & Miettinen, F. incana (Lév.) Spirin & V. Malysheva, F. infirma (Renvall & Niemelä) Miettinen & Niemelä, F. juniperina (Murrill) Spirin & Vlasák, F. kuzyana (Pilát ex Pilát) Spirin & Vlasák, F. leioderma (Mont.) Spirin & Vlasak, F. leucaena (Y.C. Dai & Niemelä) Spirin & Miettinen, F. luzonensis (Murrill) Spirin & Miettinen, F. maculatissima (Lloyd) Spirin, F. madronae (Vlasák & Ryvarden) Vlasák & Ryvarden, F. malicola (Berk. & M.A. Curtis) Spirin, F. marchionica (Mont.) Spirin & Miettinen, F. marianii (Bres.) Spirin, Vlasák & Cartabia, F. mellita (Niemelä & Penttilä) Niemelä & Miettinen, F. microcarpa (B.K. Cui & Shun Liu) Spirin, F. micropora (B.K. Cui & Shun Liu) Spirin, F. modesta (Kuntze ex Fr.) Vlasák & Spirin, F. monomitica (Yuan Y. Chen) Spirin & Viner, F. morganii (Lloyd) Spirin & Vlasák, F. moritziana (Lév.) Spirin & Miettinen, F. neotropica (D.L. Lindner, Ryvarden & T.J. Baroni) Vlasák, F. nigra (Berk.) Spirin & Miettinen, F. nivosella (Murrill) Spirin & Vlasák, F. oboensis (Decock, Amalfi & Ryvarden) Spirin, F. oleracea (R.W. Davidson & Lombard) Spirin & Vlasák, F. philippinensis (Murrill) Spirin & Vlasák, F. primaeva (Renvall & Niemelä) Miettinen & Niemelä, F. psilodermea (Berk. & Mont.) Spirin & Vlasák, F. pulverulenta (Rivoire) Rivoire, F. pulvina (Pers.) Spirin & Vlasák, F. pulvinascens (Pilát ex Pilát) Niemelä & Miettinen, F. quercina (L.) Spirin & Miettinen, F. ramentacea (Berk. & Broome) Spirin & Vlasák, F. renehenticii (Rivoire, Trichies & Vlasák) Rivoire & Vlasák, F. roseofusca (Romell) Spirin & Vlasák, F. sagraeana (Mont.) Vlasák & Spirin, F. sandaliae (Bernicchia & Ryvarden) Bernicchia & Vlasák, F. sclerotina (Rodway) M.D. Barrett & Spirin, F. serialiformis (Kout & Vlasák) Vlasák, F. serialis (Fr.) Spirin & Runnel, F. serrata (Vlasák & Spirin) Vlasák & Spirin, F. squamosella (Bernicchia & Ryvarden) Bernicchia & Ryvarden, F. stereoides (Fr.) Spirin, F. subectypa (Murrill) Spirin & Vlasák, F. substratosa (Malençon) Spirin & Miettinen, F. tropica (B.K. Cui) Spirin, F. tumulosa (Cooke) M.D. Barrett & Spirin, F. tuvensis (Spirin, Vlasák & Kotir.) Spirin & Vlasák, F. uralensis (Pilát) Spirin & Miettinen, F. ussuriensis (Bondartsev & Ljub.) Spirin & Miettinen, F. variiformis (Peck) Vlasák & Spirin, F. yunnanensis (M.L. Han & Q. An) Spirin, Daedaleopsis candicans (P. Karst.) Spirin, Megasporoporia eutelea (Har. & Pat.) Spirin & Viner, Neofomitella hemitephra (Berk.) M.D. Barrett, Pseudophaeolus soloniensis (Dubois) Spirin & Rivoire, P. trichrous (Berk. & M.A. Curtis) Vlasák & Spirin. New synonyms: Antrodia bondartsevae Spirin, A. huangshanensis Y.C. Dai & B.K. Cui, A. taxa T.T. Chang & W.N. Chou, A. wangii Y.C. Dai & H.S. Yuan, Antrodiella subnigra Oba, Mossebo & Ryvarden, Antrodiopsis Audet, Boletus quercinus Schrad., Brunneoporus Audet, Buglossoporus Kotl. & Pouzar, Buglossoporus eucalypticola M.L. Han, B.K. Cui & Y.C. Dai, Caloporus P. Karst., Cartilosoma Kotlaba & Pouzar, Coriolus clemensiae Murrill, C. cuneatiformis Murrill, C. hollickii Murrill, C. parthenius Hariot & Pat., C. rubritinctus Murrill, Daedalea Pers., Daedalea allantoidea M.L. Han, B.K. Cui & Y.C. Dai, D. americana M.L. Han, Vlasák & B.K. Cui, D. radiata B.K. Cui & Hai J. Li, D. rajchenbergiana Kossmann & Drechsler-Santos, D. sinensis Lloyd, Daedalella B.K. Cui & Shun Liu, Dentiporus Audet, Flavidoporia Audet, Fomes subferreus Murrill, Fomitopsis cana B.K. Cui, Hai J. Li & M.L. Han, F. caribensis B.K. Cui & Shun Liu, F. cystidiata B.K. Cui & M.L. Han, F. ginkgonis B.K. Cui & Shun Liu, F. iberica Melo & Ryvarden, F. incarnata K.M. Kim, J.S. Lee & H.S. Jung, F. subfeei B.K. Cui & M.L. Han, F. subtropica B.K. Cui & Hai J. Li, Fragifomes B.K. Cui, M.L. Han & Y.C. Dai, Leptoporus epileucinus Pilát, Melanoporia Murrill, Neoantrodia Audet, Neolentiporus Rajchenb., Nigroporus macroporus Ryvarden & Iturr., Niveoporofomes B.K. Cui, M.L. Han & Y.C. Dai, Pilatoporus Kotl. & Pouzar, Piptoporus P. Karst., Polyporus aurora Ces., P. durescens Overh. ex J. Lowe, P. griseodurus Lloyd, Poria incarnata Pers., Pseudoantrodia B.K. Cui, Y.Y. Chen & Shun Liu, Pseudofomitopsis B.K. Cui & Shun Liu, Ranadivia Zmitr., Rhizoporia Audet, Rhodofomes Kotl. & Pouzar, Rhodofomitopsis B.K. Cui, M.L. Han & Y.C. Dai, Rhodofomitopsis pseudofeei B.K. Cui & Shun Liu, R. roseomagna Nogueira-Melo, A.M.S. Soares & Gibertoni, Rubellofomes B.K. Cui, M.L. Han & Y.C. Dai, Subantrodia Audet, Trametes fulvirubida Corner, T. lignea Murrill, T. lusor Corner, T. pseudodochmia Corner, T. subalutacea Bourdot & Galzin, T. supermodesta Ryvarden & Iturr., T. tuberculata Bres., Tyromyces multipapillatus Corner, T. ochraceivinosus Corner, T. palmarum Murrill, T. singularis Corner, T. squamosellus Núñez & Ryvarden, Ungulidaedalea B.K. Cui, M.L. Han & Y.C. Dai. Lectotypes: Hexagonia sulcata Berk., Polyporus castaneae Bourdot & Galzin, Poria incarnata Pers., Trametes subalutacea Bourdot & Galzin, Ungulina substratosa Malençon. Neotypes: Agaricus soloniensis Dubois, Boletus pulvinus Pers. Citation: Spirin V, Runnel K, Vlasák J, Viner I, Barrett MD, Ryvarden L, Bernicchia A, Rivoire B, Ainsworth AM, Grebenc T, Cartabia M, Niemelä T, Larsson K-H, Miettinen O (2024). The genus Fomitopsis (Polyporales, Basidiomycota) reconsidered. Studies in Mycology 107: 149-249. doi: 10.3114/sim.2024.107.03.

Pseudomonas protegens volatile organic compounds inhibited brown rot of postharvest peach fruit by repressing the pathogenesis-related genes in Monilinia fructicola.

Brown rot, caused by Monilinia fructicola, is considered one of the devasting diseases of pre-harvest and post-harvest peach fruits, restricting the yield and quality of peach fruits and causing great economic losses to the peach industry every year. Presently, the management of the disease relies heavily on chemical control. In the study, we demonstrated that the volatile organic compounds (VOCs) of endophyte bacterial Pseudomonas protegens QNF1 inhibited the mycelial growth of M. fructicola by 95.35% compared to the control, thereby reducing the brown rot on postharvest fruits by 98.76%. Additionally, QNF1 VOCs severely damaged the mycelia of M. fructicola. RNA-seq analysis revealed that QNF1 VOCs significantly repressed the expressions of most of the genes related to pathogenesis (GO:0009405) and integral component of plasma membrane (GO:0005887), and further analysis revealed that QNF1 VOCs significantly altered the expressions of the genes involved in various metabolism pathways including Amino acid metabolism, Carbohydrate metabolism, and Lipid metabolism. The findings of the study indicated that QNF1 VOCs displayed substantial control efficacy by disrupting the mycelial morphology of M. fructicola, weakening its pathogenesis, and causing its metabolic disorders. The study provided a potential way and theoretical support for the management of the brown rot of peach fruits.

First Report of Brown Rot Caused by Porogramme epimiltina on Gastrodia elata in China.

Gastrodia elata Blume is a valuable medicinal plant in China with great significance in medicine (Li et al. 2023). From 2022 to 2023, G. elata tuber rot occurred in about 50 households in the main cultivation areas of G. elata (27°39' N, 104°16' E) in Yiliang County, Zhaotong, Yunnan Province, southwest China. The planting area of G. elata was 776 ha, and the incidence rate was 10%. Symptoms present as light brown lesions on the surface of the tuber, sunken, soft and foul-smelling. Infected G. elata tubers were randomly collected from each household, packed into transparent plastic bags, and strains were isolated in the laboratory as follows. The tubers of 15 infected G. elata tubers were surface-sterilized with 0.5% NaOCl for 2 min, rinsed five times with sterile water, and dried. Symptomatic tissues from the margin between necrotic and healthy tissues were cut into 5 × 5 mm pieces, placed onto potato dextrose agar (PDA), and incubated at 28 ºC in the dark for 3 days. Hyphal tips of fungi growing from the samples were transferred onto new PDA plates and incubated until they produced conidia. Two fungal strains (Charliezhao 425 and 433) with the same morphological characteristics were obtained from the samples. Colonies were whitish and grew rapidly, irregularly turning pale orange at the edge or center of the mycelium pad on a two-week-old petri dish, and finally dark red，spore oval to spherical, 2.7 to 5.3 × 2.3 to 3.5 µm (n=50). The morphological characteristics of the isolates resembled Porogramme epimiltina (Mao et al. 2023; Kubayashi et al. 2001). Genomic DNA of two representative isolates (Charliezhao 425 and 433) was extracted using the DN14 cetyltrimethylammonium bromide rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing). The ITS and TEF1 genes were amplified by polymerase chain reaction using the primers ITS1/ITS4 (White et al. 1990) and EF1-983F/EF1-2218R (Rehner et al, 2005), respectively. All sequences were deposited in GenBank (accession no. OR905803, OR905804 for ITS, OR939812, OR939813 for TEF1). A BLASTN homology search with the ITS nucleotide sequences showed that they had 98.99 to 99.15% identity with P. epimiltina isolate OP997539 (588/594 bp) and isolate OP997539 (584/589 bp), respectively; and the TEF1 sequences had 95.41 to 95.59% % identity to isolates OP556566 (540/565 bp) and isolate OP556566 (542/567 bp), respectively. To complete Koch's hypothesis, the surfaces of 5 mature and healthy G. elata tubers were disinfected with 1% NaClO solution for 1 minute, rinsed with sterile water 5 times, and dried at 25 ℃ for 30 minutes. Conidial suspensions (106 spores/ml) were collected from two isolates (Charliezhao 425 and 433) and sprayed on G. elata tuber, and the control treated with distilled water. All G. elata tubers were incubated at 25℃ with 80% relative humidity. The experiment had three replicates. After 7 days of culture, there were obvious rotten and smelly on the inoculated tubers. No symptoms were observed in the control groups. The pathogen was re-isolated from all inoculated birch tubers and confirmed as P. epimiltina by morphological and molecular analysis, which fulfilled Koch's hypothesis. Gastrodia elata is a valuable and extensively used herbal Traditional Chinese Medicine with a wide range of clinical applications. As far as we know, this is the first report of P. epimiltina causing brown rot of G. elata in China.

Regional Comparisons of Sensitivities of Phytophthora citrophthora and P. syringae Causing Citrus Brown Rot in California to Four New and Two Older Fungicides.

Isolates of the citrus brown rot pathogens Phytophthora citrophthora and P. syringae from the Inland Empire (IE) and Ventura Co. (VE) regions of southern California were evaluated for their sensitivity to ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin, and the previously published baselines that were generated for Central Valley (CV) isolates of California were expanded. Fungicides were generally more toxic to CV isolates of both species for all four fungicides. Specific differences were found in the toxicity of ethaboxam to P. syringae where CV isolates on average were 6.8 or 8.2 times more sensitive than those from the VE or IE regions, respectively. Based on the grouping of isolates in an unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, as well as fastStructure analyses and plotting of principal component analyses (PCAs), differences in ethaboxam sensitivity could be related to differences in genetic background of the isolates. Isolates of P. citrophthora from the IE and VE had slightly reduced (i.e., 1.5×) sensitivity to mandipropamid as compared with isolates from the CV and were found on distinct branches in the UPGMA dendrogram. Differences in genetic background of less sensitive isolates within each species indicate that these two phenotypes emerged multiple times independently. IE and VE isolates of both species were sensitive to mefenoxam. Moderate resistance to potassium phosphite (EC50 values of 25 to 75 µg/ml) was present in IE and VE isolates of P. syringae, whereas some IE isolates of P. citrophthora were considered resistant with EC50 values of up to 113.69 µg/ml. Resistance to potassium phosphite did not relate to distinct genotypes.

Comparative fitness of Monilinia fructicola isolates with multiple fungicide-resistant phenotypes.

This study characterized 52 isolates of Monilinia fructicola from peach and nectarine orchards for their multi-resistance patterns to thiophanate-methyl (TF), tebuconazole (TEB), and azoxystrobin (AZO) using in vitro sensitivity assays and molecular analysis. The radial growth of M. fructicola isolates was measured on media amended with a single discriminatory dose of 1 µg/ml for TF and AZO and 0.3 µg/ml for TEB. Cyt b, CYP51, and ß-tubulin were tested for point mutations that confer resistance to quinone outside inhibitors (QoIs), demethylation inhibitors (DMIs), and methyl benzimidazole carbamates (MBCs), respectively. Eight phenotypes were identified including isolates with single, double, and triple in vitro resistance to QoI, MBC, and DMI fungicides. All resistant phenotypes to TF and TEB presented the H6Y mutation in ß-tubulin and the G641S mutation in CYP51. None of the point mutations typically linked to QoI resistance were present in the Monilinia isolates examined. Moreover, fitness of the M. fructicola phenotypes was examined in vitro and detached fruit assays. Phenotypes with single-resistance displayed equal fitness in in vitro and fruit assays compared to the wild-type. In contrast, the dual and triple-resistance phenotypes suffered fitness penalties based on osmotic sensitivity and aggressiveness on peach fruit. In this study, multiple resistance to MBC, DMI, and QoI fungicide groups was confirmed in M. fructicola. Results suggest that Monilinia populations with multiple resistance phenotypes are likely to be less competitive in the field than those with single resistance, thereby impeding their establishment over time and facilitating disease management.

Streptomyces virginiae XDS1-5, an antagonistic actinomycete, as a biocontrol to peach brown rot caused by Monilinia fructicola.

BACKGROUND: Peach brown rot, caused by the pathogen Monilinia fructicola, represents a significant postharvest infectious disease affecting peach fruit. This disease is responsible for a substantial increase in fruit decay rates, leading to significant economic losses, often exceeding 50%. Currently, there is a growing interest in identifying biocontrol agents to mitigate peach brown rot, with a predominant interest in Bacillus species. RESULTS: In this investigation, we isolated 410 isolates of actinomycetes from non-farmland ecosystem soil samples. Subsequently, 27 isolates exhibiting superior inhibitory capabilities were selected. Among these, strain XDS1-5 demonstrated the most robust fungistatic effect against brown rot disease, achieving an 80% inhibition rate in vitro and a 66% inhibition rate in vivo. XDS1-5 was identified as belonging to the Streptomyces virginiae species. Furthermore, a fermentation filtrate of XDS1-5 exhibited the ability to metabolize 34.21% of the tested carbon sources and 7.37% of the tested nitrogen sources. Particularly noteworthy was its capacity to disrupt the cell membrane structure directly, leading to increased cell membrane permeability and cytoplasmic leakage. Additionally, our investigation indicated that indoline, a metabolite produced by XDS1-5, played a pivotal role in inhibiting the growth of M. fructicola. CONCLUSION: In summary, our study has identified a biocontrol actinomycete, XDS1-5, with the potential to effectively inhibit postharvest brown rot disease in peaches. This finding holds great significance for the biological control of peach brown rot, offering promising prospects for mitigating the economic losses associated with this devastating disease. © 2024 Society of Chemical Industry.

Pectin decomposition at the early stage of brown-rot decay by Fomitopsis palustris.

The sapwood of Japanese cedar (Cryptomeria japonica D. Don) was decayed by the brown-rot fungus Fomitopsis palustris under bright and dark conditions. Scanning electron microscopy revealed the presence of mycelia inside the wood even after 1 week from the start of fungal exposure. Moreover, holes were observed in the torus after fungal exposure. Ruthenium red staining revealed that the pectin in pits was largely absent for 3 weeks. These events occurred before the mass loss of wood samples was confirmed at the early stage. Moreover, FpPG28A was more highly expressed at the hyphal front on a pectin-containing medium under dark conditions compared with bright conditions. This up-regulation under dark conditions indicated that the pectin decomposition ability was promoted inside the wood where light could not reach. In conclusion, we suggest that the brown-rot fungus completed its hyphal expansion within the wood via pectin decomposition in pits before holocellulose decomposition.

Low frequency of resistance to thiophanate-methyl in Monilinia fructicola populations from southeastern United States peach orchards.

Methyl benzimidazole carbamate (MBC) fungicides were once widely used for brown rot (Monilinia fructicola) control of peach (Prunus persica (L.) Batsch) in the southeastern US, but their use was substantially reduced and often eliminated due to widespread resistance. In this study, 233 M. fructicola isolates were collected from major peach production areas in Alabama, Georgia, and South Carolina, and sensitivity to thiophanate-methyl was examined. Isolates were also collected from one organic and two experimental peach orchards. A discriminatory dose of 1 µg/ml was used to distinguish sensitive (S) and moderately sensitive (S-LR) isolates from low resistant phenotypes, while 50 and 500 µg/ml thiophanate-methyl concentrations were used to determine high resistant (HR) phenotypes. Sequence analyses were performed to identify mutations in the ß-tubulin target gene and detached fruit assays were performed to determine the efficacy of a commercial product against isolates representing each phenotype. Results indicated 55.7%, 63.5%, and 75.9% of isolates from Alabama, Georgia, and South Carolina, respectively, were S to thiophanate-methyl; 44.3%, 36.5%, and 21.4% were S-LR; no isolates were LR; and only 3 isolates (1.3%) from South Carolina were HR. No mutations in S or S-LR isolates were found, but HR isolates revealed the E198A mutation, an amino acid change of glutamic acid to alanine conferring high resistance. The high label rate of a commercial product containing thiophanate-methyl controlled brown rot caused by S and S-LR isolates in detached fruit studies but was ineffective against HR isolates. The combinations of thiophanate-methyl with azoxystrobin or isofetamid, when mixed together and applied in an experimental orchard 14 days preharvest, significantly reduced brown rot incidence on pre and postharvest commercially ripe fruit and efficacy was comparable to that of a grower standard fungicide. These results indicate that thiophanate-methyl may again be useful to peach growers in the southeastern US for brown rot and fungicide resistance management.

Effect of L-cysteine treatment to induce postharvest disease resistance of Monilinia fructicola in plum fruits and the possible mechanisms involved.

Plum is an important stone fruit in China, but the fruit is easily perishable and susceptible to infection by pathogens. Traditionally, synthetic fungicides are used to control diseases. However, the side effects of fungicides should not be ignored. Cysteine, generally recognized as safe (GRAS) amino acid, has been reported to play roles in the plant abiotic stress response, but little is known about the role of cysteine to control postharvest diseases in fruits. Therefore, this study was designed to investigate the effect of L-cysteine treatment on control of postharvest brown rot in artificially inoculated plum fruits and the possible biocontrol mechanisms involved. Postharvest plum fruits were inoculated with 1, 10, 100 and 1000 mg L-1 L-cysteine. 100 mg L-1 L-cysteine treatment effectively controlled brown rot in artificially inoculated plum fruits by inducing resistance. Furthermore, 100 mg L-1 L-cysteine treatment increased the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), enhanced the content of NADPH of the pentose phosphate pathway, as well as improved the contents of H2O2 and some amino acids in the artificially inoculated plum fruits. 100 mg L-1 L-cysteine treatment also elevated the antioxidant content (AsA, GSH) and the antioxidant enzymes activities (APX, GR, MDAR, DHAR) of the ascorbate-glutathione (AsA-GSH) pathway. The protective effects of L-cysteine treatment on postharvest plum fruits likely be due to activating some defense-related responses of the fruit against infection. L-cysteine treatment is a safe promising method for controlling postharvest brown rot in plum fruits.

First Report of Lasiodiplodia theobromae Causing Stem Brown Rot of Loquat in China.

Loquat (Rhaphiolepis biabas, heterotypic synonym: Eriobotrya japonica) is an important edible and medicinal plant that is widely cultivated on 133 thousand hectares (recorded in 2022) in China. A stem brown rot was observed on young and old trees in Mengzi city (23°23' N; 103°23' E), Yunnan Province, southwest China, during October 2014 and September 2021. Incidence ranged from 20% of trees in surrounding plantations to 50% incidence of a 160 tree orchard that was the focal point of the disease survey. Circular brown lesions occurred initially on the stems and gradually covered all the epidermis of the stem, leading to irregular dents within the bark that developed a dark brown powdery appearance (Fig.1A). Larger lesions affected vascular tissues, causing diseased trees to wither and die. Diseased tissues were surface-disinfected in a 5% sodium hypochlorite solution for 3 min, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), and incubated in the dark at 28°C. Twenty samples were collected for tissue isolation, and 11 isolates were single-spored on water agar. In culture, the colonies on PDA were white to dark-gray, velvet, with dense hyphae, diameter 7.64 cm after 5 days. After 18 days, spherical or subglobose pycnidia were developed and semi-buried in medium, their walls were thicker and dark-brown, which were black particles surrounded by gray-black hyphae. Conidiogenous cells were hyaline, cylindrical, holoblastic, slightly swollen at the base, with rounded apex. Conidia were initially hyaline and aseptate with elliptic or ovate shape, becoming dark brown with a single septate and developing longitudinal striations along thick walls at maturity. Conidia dimensions varied from 8.0 to 12.2 × 3.8 to 6.1µm (n=50) (Fig.1D). The morphological characteristics of eleven isolates were consistent with the description of Lasiodiplodia theobromae (Alves et al. 2008). Further confirmation was also determined by sequencing the internal transcribed spacer (ITS), ß-tubulin genes, partial translation elongation factor-1α (TEF-1α) (White et al. 1990, Carbone et al. 1999, Glass et al.1995). The isolate LSB-1 was selected for DNA sequence analysis. Based on BLASTn analysis, ITS sequences (OM617921) had 98.3% similarity with L. theobromae CBS164.96 (accession AY640255), CBS124.13(accession DQ458890), CAA006 (accession DQ458891) and CBS111530 (accession EF622074), ß-tubulin sequences (OM643838) showed 99.1% similarity with L. theobromae accessions EU673110. The TEF-1α (OM643839) had 99.0% identity with L. theobromae accession EF633054. The isolate LSB-1 clustered on the same clade with other L. theobromae. Pathogenicity testing of isolate LSB-1, LSB-2, LSB-3 was conducted by inoculating the stems of l-year-old seedlings growing in pots. The epidermis at the inoculation site, 15-20 cm below the crown, was wiped with 75% alcohol cotton ball, washed three times with sterile water, and then punctured (5mm diameter) with sterile inoculation needle. A 5mm block of each isolate cultured on PDA for seven days was attached to the inoculation site. Controls were inoculated with sterile PDA blocks. The inoculation area was covered with polyethylene cling film. All inoculated seedlings were kept in controlled greenhouse at 27°C with 80% relative humidity under natural daylight conditions, and watered weekly. Each treatment was repeated three times. Eight days after inoculation, all diseased plants showed dark brown discoloration at the point of inoculation (Fig. 1G) with the bark at the inoculation site gradually raising as the disease progressed. Thirty days after inoculation, all inoculated seedlings produced typical symptoms, whereas the control seedlings remained healthy. Fungal isolates were only recovered from symptomatic stems and were morphologically identical to L. theobromae, completing Koch's postulates. According to the relevant literature, Lasiodiplodia theobromae has a broad host range, causing numerous diseases, including canker and dieback of branch (Aguilera-Cogley et al., 2021), panicle blight (Mahadevakumar et al, 2022), root rot (Abd-El Ghani and Fatouh, 2005), fruit rot(Freire et al., 2011) in diverse geographical regions. To our knowledge, this is the first report of L. theobromae causing stem brown rot of loquat in China and provides a foundation for further study of the epidemiology and integrated management of this disease.

First Report of Brown Rot Caused by Monilia yunnanensis on Sweet Cherry in China.

Chinese cherry industry has developed rapidly over the past few years, with the planting acreage continuously expanding, from Shandong province to Liaoning, Shaanxi, Hebei, Sichuan etc. Monilia spp. are the most important causal agents of brown rot of cherry, to date, M. fructicola, M. mumecola, and M. fructigena were reported to cause brown rot of cherry in China (Chen et al. 2013; Yin et al. 2014; Liu et al. 2012). In May 2023, fruit of sweet cherry cultivar 'Hongdeng' (Prunus avium L.) with symptoms resembling brown rot were collected from Tongchuan City, Shaanxi Province. Conidia on diseased tissues were spread on a water agar (WA, 1.5% agar and distilled water) medium and isolated with a glass needle under a professional single spore separation microscope (Wuhan Heipu Science and Technology Ltd., Wuhan, China). If no conidia were present, fruit pieces (5 × 5 mm) at the intersection of healthy and diseased tissues were surface sterilized with a sodium hypochlorite solution (1%) for 30 s and washed three times in sterilized water, followed by 75% ethanol for 30 s, then washed three times in sterilized water. After the tissue pieces were dried, they were placed on potato dextrose agar (PDA; 200 g of potato, 20 g of dextrose, and agar at 20 g/L) and incubated at 22 °C for about twenty days to produce spores and then single spore isolation was carried out. Thirty single-spore isolates were obtained and all were morphologically similar. The isolates produced white-gray colonies with even margins and concentric rings of sporogenous mycelium after 3 days incubation, and abundant black-colored stromata on the PDA medium after 15 days of incubation at 22°C. Conidia were one-celled, hyaline, ellipsoid to lemon shape (14.12 × 10.37 µm), with 1-2 germs which is similar to M. yunnanensis on peach. The genomic DNA of the isolates was extracted as described previously (Chi et al. 2009). The pathogen identity was confirmed by multiplex PCR which resulted in a 237bp amplicon, which is diagnostic of M. yunnanensis (Hu et al. 2011). Further sequencing of the internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene (accession number: OR192774) indicated 100% identity with that of M. yunnanensis isolates (accession numbers: MW355895, ON024742). The average daily growth of mycelium on PDA at 22°C was 11.44 mm. Koch's postulates were fulfilled by inoculating 20 mature sweet cherry fruits of cv. 'Van' with mycelial plugs in a drilled hole. After 3 days of incubation at 22℃ in an airtight plastic tray with wet paper, the inoculated fruit developed typical brown rot symptoms. The developing spores on inoculated fruit were confirmed to be M. yunnanensis based on ITS sequence. All control fruit inoculated with a PDA plug remained healthy. M. yunnanensis was first reported as the causal agent of brown rot of peach in China (Hu et al. 2011). Later studies demonstrated that it is also pathogen on other fruits, e.g. hawthorn (Zhao et al. 2013), plum (Yin et al. 2015), apricot (Yin et al. 2017), apple, and pear in China (Zhu et al. 2016). To our knowledge, this is the first report of cherry brown fruit rot caused by M. yunnanensis, indicating the high risk of this species to cherry production, and effective strategies must be taken to prevent the possible control failure in practice.

First Report of Brown Rot Disease on Cultivated Auricularia cornea Caused by Trichoderma pleuroticola in Sichuan Province, China.

Auricularia cornea Ehrenb. is a well-known, rare, and valuable edible mushroom, with considerable culinary and medicinal value. It is distributed worldwide and especially common in Asia (Khatua et al. 2022). In China, more than 1.89 million tons of A. cornea are artificially cultivated annually, particularly in Sichuan Province, which produces 0.97 million tons, accounting for about 51% of the nation's total (Ye et al. 2022). However, farmers in Shifang, a county-level city in Sichuan Province, who practiced traditional greenhouse cultivation on a large scale, reported that brown rot disease affected up to 20% of their A. cornea crop and caused severe failures each year between 2016 and 2022, resulting in devastating economic losses. Worse, when the diseased fruit bodies were not removed promptly by farmers, the disease could spread to 100% of crop in a given greenhouse in 10 to 15 days. The symptoms mainly occur in fresh unfolded fruiting bodies. Lesions were brown, putrefied, and foul smelling, and eventually the fruiting bodies wilted. Naturally air-dried fruiting bodies were dark brown with yellow deposits at the edges (Fig. 1A). In this backdrop, we collected more than 60 diseased samples from Shifang (104°1'15''E, 31°12' 30'' N) and isolated pathogens from May 2021 through May 2022. On the clean bench, the surfaces of the lesions were disinfected with cotton balls soaked in 75% ethanol and rinsed three times with sterile water, and then the internal tissue block was picked with a sterile scalpel and cultured in potato dextrose agar (PDA) medium at 25°C. The pathogen was repeatedly isolated and purified, and we conducted pathogenicity tests. Colonies of the pathogen on PDA medium were white and cottony, with a mycelial growth rate of 13.92 ± 1.24 mm/day at 25°C. Then the spores began to turn yellow-green and soon turquoise, converging into a wide concentric wheel. The spores were elliptical with dimensions of 4.0 to 8.0 µm × 3.0 to 4.5 µm (N=50). Pathogenicity tests were conducted in an incubator. Ninety pure white, healthy A. cornea fruiting bodies were randomly picked and placed in groups of 10. On the clean bench, the bodies surface were sterilized with 75% alcohol cotton balls, then washed three times with sterile water, dried with sterile absorbent paper, and placed in sterile petri dishes for subsequent tests. The first control group (CK1) did not receive additional treatment other than the surface disinfection mentioned above. These bodies were immediately sealed with parafilm. The second control group (CK2) was not inoculated with the pathogen, and a blank sterile PDA plug (diameter: 0.8 cm) was placed on the surface of the fruiting body and sealed with parafilm. All seven treatment groups (TS) were inoculated with single-pathogen mycelium plugs (diameter: 0.8 cm) in the center surface of each fruiting body. They were then sealed with parafilm. All total of nine experimental treatment groups were cultured in a light incubator at 25°C. The test results showed that the pathogen could visibly infect the A. cornea within 24 hours. At 48 hours after inoculation, the lesions were round and brown, radiating outward along the inoculum, consistent with the symptoms of the original sample. As culture time continued, the extent of each lesion gradually expanded (Fig. 1B). After 120 hours, the fruiting bodies showed rot, stench, and loss of other traits relevant to commercial value. In contrast, the CK1 and CK2 groups had no lesions. Furthermore, the fungal cultures with the same phenotypic characteristics could be continuously isolated from the lesions of TS, and the pathogenic factors were verified by Koch's postulates. Similarly, uninoculated and inoculated tests were conducted in a greenhouse, and the results were consistent with those of incubator testing. The pathogen was designated MMEBYJ202206. The DNA of the pathogen was extracted using CTAB, and the rDNA internal transcribed spacer (ITS) of the isolates was amplified using ITS1/ITS4 primers. PCR was conducted in a 25 µL reaction mixture, and a 596 bp sequence was obtained by sequencing. The ITS sequence has been submitted in GenBank with accession number ON974844.1. BLAST database in NCBI was used to compare ITS sequences and phylogenetic tree was constructed based on the neighbor-joining algorithm from MEGA (Fig. 2). The results indicate that the MMEBYJ202206 was located on a common clade of the phylogenetic tree with KX343129.1, KX343130.1, KX343131.1, and MF871554.1 of T. pleuroticola, and it showed 99% support in bootstrap (500 replicates), but it was not in the same clade as other Trichoderma spp., suggesting that the pathogen was T. pleuroticola. To our knowledge, this is the first report to show that T. pleuroticola can cause brown rot disease on artificially cultivated A. cornea. However, a previous study showed T. pleuroticola to be the causal agent of considerable decline in the yield of Pleurotus ostreatus and P. florida (Siwulski et al. 2011; Blaszczyk et al. 2013) and capable of infecting A. heimuer raised on artificial bed-log (a rod used for growing mycelium of edible mushrooms) (Liu et al. 2019). Brown rot disease is important in China because it has caused considerable damage to yield in artificially cultivated A. cornea, a decline in the external and internal qualities of the product, and a reduction in the enthusiasm of farmers for this crop. Consequently, this study provides a foundation for further research and prevention of this pathogen in China.

First Report of Bacterial Brown Rot Disease Caused by Ewingella americana on Cultivated Naematelia aurantialba in China.

Naematelia aurantialba (synonym Tremella aurantialba) is one of the jelly fungi and highly valued edible and medicinal mushrooms. It has been cultivated industrially in recent years and consumed popularly in China. In September 2022, brown rot disease of fruiting bodies was observed at the N. aurantialba factory in Tongzhou district, Beijing with a disease incidence of ~10%. Symptoms initially appeared as color changing from orange to light brown. The infected area expanded gradually until covered fully the fruiting body. Meanwhile, the interior of the fruiting body became rotten and dark brown. Finally, the whole fruiting body became wrinkled and brown, resulting in significantly reduced yield and economic loss. Isolations were made from 12 infected mushroom samples. Infected tissue within the fruiting body was mashed in sterilized 1.5 mL tubes containing 1 mL of sterile distilled water. After standing for 5-10 min, the suspensions were streaked on the Luria-Bertani (LB) medium and cultured at 37°C for 24h. The physiological and biochemical reactions of isolated strains were determined using the API 20E system (Reyes et al. 2004) according to the manufacturer's instructions. All the strains showed the same reaction results. The bacterial colonies were streaked on fresh LB medium at 37°C for 24 h, and a single pure culture was obtained with round, smooth and semitransparent. The bacterial cells were gram-negative, short-rod, (0.3) 0.8-2.0 (2.5) × (0.1) 0.6-1.0 (1.5) µm, and peri-flagellate. The isolates were further confirmed by sequence analysis of the 16S rRNA and gyrB genes with primer 27F/1492R and gyrB-UP1s/gyrB-UP2sr (Liu et al. 2018). Using EzBioCloud data searches, the 16S rRNA sequence of four strains (GenBank accession OP727593, OP727595, OP727596, OP727601) matched the sequence of E. americana type strain ATCC 33852 (accession JMPJ01000013) with identity of 99.65%~99.93 and 100% completeness. The GyrB sequence matched the E. americana in GenBank (MK460250) and showed 98.71% identity and 100% completeness. Finally, the pathogen was identified as E. americana based on morphological, physiological, biochemical, and molecular characteristics. The pathogenicity test was conducted by spreading bacterial suspensions cultured 48h onto 12 healthy cultivated fruiting bodies of N. aurantialba, with sterile distilled water as a control, and then cultured in a chamber at 23°C with 85% relative humidity. Brown symptoms, similar to natural symptoms, were observed on all inoculated fruiting bodies after 48h, whereas the controls remained symptomless. Pathogenic bacteria were isolated from the inoculated fruiting body and confirmed to be E. americana based on morphological and 16S rRNA molecular characteristics, thus fulfilling Koch's postulates. E. americana caused stipe necrosis on Agaricus bisporus in Egypt (Madbouly et al. 2014), the oak tree in Thailand, and pneumonia in Humans (Doonan et al. 2016), and brown blotch on Flammulina velutipes (Liu et al. 2018). To our knowledge, this is the first worldwide report of E. americana infecting jelly fungus N. aurantialba causing brown rot disease. E. americana is an opportunistic cross-kingdom pathogen (Liu et al. 2018). That will provide a critical alert on the prevention, effective monitoring, and control of the disease.

First Report of Apple Brown Rot caused by Monilinia polystroma in Jilin Province in China.

Brown rot caused by Monilinia spp. is an important postharvest disease. It affects fruit quality and can cause serious economic losses. In October 2021, typical brown rot symptoms on fruit were observed at an apple orchard in Xiaobaishan Township, Jilin Province, China (E126°39'10″, N43°44'21″). Over 1200 plants were surveyed in the orchard, and nearly 25% of the plants were infected. In this research, samples from ten different trees showing typical symptoms were isolated and identified. Freshly diseased fruits were surface sterilized with 75% ethanol for 15s, then fungal colonies were isolated from 3 mm diameter diseased tissue samples. The purified colonies were placed on potato dextrose agar (PDA), oatmeal agar (OA) and water agar (WA + Sterilized apple pulp) and incubated at 25 ℃ in a 12 h/12 h light-dark photoperiod for 5 days. The colonies became light to dark brown; they grew faster on PDA with a growth rate of 5.53 mm/d, most densely on OA and slowest on SA + sterilized apple pulp with thin mycelia. After 20 days, some transparent, oval, smooth conidia were observed on the SA + sterile apple culture medium. Conidia sizes were 8.27-16.54 (avg. 11.97) x 2.92-7.09 (avg. 4.52) um (n=50) (Hilber-Bodmer et al. 2012). Pure cultures of eight samples were isolated from single spores, and DNA was extracted with a commercial nucleic acid extraction kit (Omega, cat#D3390-01). Then, fragments of the internal transcribed spacer region (ITS), translation elongation factor 1 alpha (EF1-alpha), beta-tubulin (Bt), and glyceraldehyde-3-phosphate dehydrogenase genes(G3pdh) were PCR amplified using primes ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), BT-2a/BT-2b (Glass and Donaldson 1995), and G3pdhF/G3pdhR (Hu et al. 2011) respectively. The amplicons were sequenced by Takara Bio Inc. Sequences of all isolates were identical, thus one set of sequences were run with BLASTn against the NCBI GenBank nr database. The homology analysis showed that ITS (OQ170786), Bt (OQ179019), EF (OQ834046) and G3pdh (OQ834047) gene fragments were 100% (0/540 nt) , 100% (0/462 nt), 99.01% (3/304nt) and 99.61% (3/767 nt) similar to M. polystroma （NR154198.1, MT038414.1, LT632542.1, MT038415.1）respectively. Based on morphological characteristics and sequence analysis, the fungal isolate was identified as M. polystroma. Koch's postulates were conducted using ten healthy apple fruits that were surface disinfected with 0.2% sodium hypochlorite and repeatedly rinsed with sterile water. The test apples were wounded with a sterile needle, inoculated with mycelial agar plugs (3 mm diameter) on the wounds, and incubated at 25℃, 50% room humidity. The equivalent sterile PDA plugs were used as control and the experiment was repeated three times. After 5 days, brown rot symptoms appeared on the inoculated apples. 10 days later, nearly 1/3 of the inoculated apple surface appeared rotted, but the controls were symptomless. Subsequently, the same strain was re-isolated from the inoculated apples by pure culture and molecular identification. Therefore, M. polystroma (named JL-1) was confirmed as the causal agent of brown rot in Jilin Provincen China. M. polystroma is a typical pathogen of brown rot in the north of China, and only reported on apples in Shandong, apricots in Heilongjiang and pears in Hebei in China (Zhu et al. 2016) but never in Jilin. In addition, it was reported that the contribution of M. polystroma species to brown rot disease on apple and pear in China is 20% out of all the Monilinia spp. species that cause the disease, but M. polystroma virulence is not significantly different from other Monilinia species more widely distributed (Zhu et al, 2016). This is the first report of M. polystroma causing apple brown rot in Jilin Province of China. This finding will provide useful information for future diagnosis and management.

First Report of Brown Rot Caused by Monilinia polystroma on Sweet Cherry and Almond in Italy.

Brown rot decay is an important disease of pome and stone fruits. In Italy, the main pathogens on stone fruits are Monilinia laxa, M. fructigena, and M. fructicola (Spitaler et al. 2022a). In addition, Monilinia polystroma (G. Leeuwen) L. M. Kohn (van Leeuwen et al. 2002), was recently found in Italy on peach (Martini et al. 2014), pear (Martini et al. 2015), plum (Abate et al. 2018), apple (Rosati et al. 2021), and quince (Spitaler et al. 2022b). In South Tyrol province, sweet cherry (Prunus avium L.) and almond (Prunus dulcis Mill. D. A. Webb), plants of the Rosaceae family and belonging to stone fruits, were observed to be frequently affected by brown rot. Affected cherries as well as almonds showed brown lesions, covered by yellowish or buff-colored stroma in concentric rings. Symptomatic cherries became shriveled, while symptomatic almonds remained firm. To determine the pathogen, single spore isolates were obtained from five symptomatic fruits, each from a cherry orchard of the cultivar Kordia in July 2021 and almond trees of the variety Dulcis in August 2021. Both sample sites were situated in Vadena/Pfatten. Infestation in the cherry orchard, covered by a rain-protection foil, was determined to be about 1 %. In almond, over 50 % of the fruits of various ripening stages showed brown rot symptoms. On potato dextrose agar (PDA) at 22 °C and a photoperiod of 16 h, isolates from both fruits matched the morphological characteristics of M. polystroma (Vasic et al. 2016) within 14 days. DNA was extracted from mycelium and the rRNA encoding gene region using ITS4 and ITS6 primers as well as a genomic sequence of unknown function using the primers UniMon_Forw and UniMon_Rev (Petróczy et al. 2012), were amplified and sequenced. MegaBLAST analysis revealed 100 % identity with M. polystroma sequences of the NCBI GenBank (rRNA encoding region: NR_154198; genomic region: JN128836). Sequences were deposited in GenBank under the accession numbers OP642545/OP654171 (cherry) and OP642546/OP654172 (almond). Pathogenicity was confirmed with mature cherries cultivar Duroncino or almost mature almond fruit of the variety Dulcis, respectively: 16 samples each for both fruits were surface-sterilized by dipping in 75 % ethanol for 10 s and subsequent rinsing with sterile water for 10 s. Mycelial plugs (1 mm) were dislodged from a 7-day old colony and inserted in a 1 mm hole into the fruits. Incubation was performed in plastic boxes under the conditions described above. PDA-inoculated fruit were used as controls. All cherries and all almonds were completely covered by brown rot lesions 7 days and 15 days post inoculation, respectively. Control fruits remained symptomless. Conidia were produced in branched chains on mycelium-inoculated fruit. Conidia were one-celled, limoniform, hyaline, measuring 13.1 to 22.2 × 9.7 to 14.8 µm (cherry) and 14.1 to 20.8 × 10.7 to 15.3 µm (almond). Additionally, 16 fruits each were inoculated with 20 µL conidial suspension (5 x 10^3 spores/mL) from mycelium-inoculated fruits. All cherries as well as all almonds were completely covered by brown rot lesions 7 days and 15 days post inoculation. Control fruits remained symptomless. To confirm identity, the fungus was isolated from five spore-inoculated fruits each for cherry and almond. The isolates showed identical morphological features and sequence identity as the original isolates. To our knowledge, this is also the first report of M. polystroma on almond, while the pathogen has already been reported on sweet cherry in Poland (Poniatowska et al. 2016). These additional host plants identified in this study suggest a broad impact of M. polystroma on Italian stone fruit production. Due to the economically important cultivation of stone fruit, further knowledge about the pathogens' host range will help to assign brown rot symptoms to M. polystroma and to improve targeted control strategies.

Molecular and Morphological Identification of Monilia yunnanensis Causing Brown Rot on Chinese Quince and Peach in Yunnan, China.

More than 30% of fruits of Chinese Quince (Chaenomeles speciosa) and peach (Prunus persica) showed circular, water-soaked and brown spots in July 2022 in Kunming, Yunnan, China. The center of these spots was covered by a large number of earthy brown and oblate sporogeneous mycelium containing conidiophore and conidia, which were one-celled, limoniform, hyaline (13.73 to 22.77 x 8.17 to 12.84 µm, n=50). By September 2022, almost 100% of fruits showed symptoms. Later, most of them fell or a few stiff, black and mummified fruits were left on the trees. Fungal isolates were isolated by single-spore technique on Potato Dextrose agar (PDA) from the diseased fruits, and incubated at room temperature (20-28 °C) in darkness for 14 days. The colony was gray, smooth at margins, 7.6-8.0 cm in diameter. To fullfill Koch's postulates, mycelial plugs of one representative isolate YHD611 from Chinese Quince and another YHD610 from peach were used to inoculate three wounded and three non-wounded surface-disinfected fruits of both hosts at room temperature (19-27 °C), respectively. Three wounded and three non-wounded fruits inoculated with sterile PDA plugs served as the control. The wounded peaches appeared water-soaked and had brown lesions after three days of inoculation, then completely decayed after nine days, while non-wounded fruits showed symptoms after five days. The wounded fruits of Chinese Quince developed similar symptoms after eight days of inoculation, and completely decayed after 13 days, while non-wounded fruits showed obvious symptoms after 15 days. In a subsequent study, isolate YHD611 was inoculated to peach while isolate YHD610 was inoculated to Chinese Quince to understand host specificity of the isolates. The results showed that when peaches were infected with YHD611, symptoms were observed on wounded fruits after three days while on non-wounded fruits after five days. When Chinese Quince was infected with YHD610, symptoms were observed on wounded fruits after 14 days while on non-wounded fruits after 21 days. Fungal isolates from symptomatic fruits were identical to the original isolates. There were no symptoms on the control fruits of both hosts. Molecular identification was confirmed based on the sequences of internal transcribed spacer (ITS, primers ITS1 and ITS4) and ß-tubulin (TUB2, primers Bt2a and Bt2b) genes (Niu et al. 2016). BLASTn analysis of the ITS (OQ15519and OQ155196) and TUB2 (OQ185202 and OQ185201) of YHD611 and YHD610 revealed a 100% sequence identity, respectively, to Monilia yunnanensis AH7-2 (KT735924.1 for ITS, KT736008.1 for TUB2). In the phylogenetic analyses based on ITS and TUB2 sequence data, the isolates YHD611 and YHD610 belonged to the M. yunnanensis clade. Based on morphological and molecular identification, both isolates were identified as M. yunnanensis, which was reported as the pathogen causing brown rot of plum, peach, apple and pear in Yunnan, China (Hu et al. 2011; Yin et al. 2015). To our knowledge, this is the first report of M. yunnanensis causing brown rot on the fruits of Chinese Quince in Yunnan, China. This study also reports that M. yunnanensis from Chinese Quince can infect peach, and the pathogen from peach can infect Chinese Quince. These findings suggest that M. yunnanensis can transfer from one host to another and causing serious economic losses in multiple fruit crops in Yunnan, China. References: Hu, M. J., et al. 2011. PLoS One. 6:e24990. Niu, C. W., et al. 2016. Mycosystema, 35(10):1. Yin, L. F., et al. 2015. Plant Dis. 99:1775.

Draft Genome and Biological Characteristics of Fusarium solani and Fusarium oxysporum Causing Black Rot in Gastrodia elata.

Gastrodia elata is a valuable traditional Chinese medicinal plant. However, G. elata crops are affected by major diseases, such as brown rot. Previous studies have shown that brown rot is caused by Fusarium oxysporum and F. solani. To further understand the disease, we studied the biological and genome characteristics of these pathogenic fungi. Here, we found that the optimum growth temperature and pH of F. oxysporum (strain QK8) and F. solani (strain SX13) were 28 °C and pH 7, and 30 °C and pH 9, respectively. An indoor virulence test showed that oxime tebuconazole, tebuconazole, and tetramycin had significant bacteriostatic effects on the two Fusarium species. The genomes of QK8 and SX13 were assembled, and it was found that there was a certain gap in the size of the two fungi. The size of strain QK8 was 51,204,719 bp and that of strain SX13 was 55,171,989 bp. Afterwards, through phylogenetic analysis, it was found that strain QK8 was closely related to F. oxysporum, while strain SX13 was closely related to F. solani. Compared with the published whole-genome data for these two Fusarium strains, the genome information obtained here is more complete; the assembly and splicing reach the chromosome level. The biological characteristics and genomic information we provide here lay the foundation for further research on G. elata brown rot.

Distinctive carbon repression effects in the carbohydrate-selective wood decay fungus Rhodonia placenta.

Brown rot fungi dominate the carbon degradation of northern terrestrial conifers. These fungi adapted unique genetic inventories to degrade lignocellulose and to rapidly release a large quantity of carbohydrates for fungal catabolism. We know that brown rot involves "two-step" gene regulation to delay most hydrolytic enzyme expression until after harsh oxidative pretreatments. This implies the crucial role of concise gene regulation to brown rot efficacy, but the underlying regulatory mechanisms remain uncharacterized. Here, using the combined transcriptomic and enzyme analyses we investigated the roles of carbon catabolites in controlling gene expression in model brown rot fungus Rhodonia placenta. We identified co-regulated gene regulons as shared transcriptional responses to no-carbon controls, glucose, cellobiose, or aspen wood (Populus sp.). We found that cellobiose, a common inducing catabolite for fungi, induced expression of main chain-cleaving cellulases in GH5 and GH12 families (cellobiose vs. no-carbon > 4-fold, Padj < 0.05), whereas complex aspen was a universal inducer for Carbohydrate Active Enzymes (CAZymes) expression. Importantly, we observed the attenuated glucose-mediated repression effects on cellulases expression, but not on hemicellulases and lignin oxidoreductases, suggesting fungi might have adapted diverged regulatory routes to boost cellulase production for the fast carbohydrate release. Using carbon regulons, we further predicted the cis- and trans-regulatory elements and assembled a network model of the distinctive regulatory machinery of brown rot. These results offer mechanistic insights into the energy efficiency traits of a common group of decomposer fungi with enormous influence on the carbon cycle.