Involvement of the Autophagy Protein Atg6 in Development and Virulence in the Gray Mold Fungus Botrytis cinerea

Gray mold caused by Botrytis cinerea is a devastating disease that leads to huge economic losses worldwide. Autophagy is an evolutionarily conserved process that maintains intracellular homeostasis through self-eating. In this study, we identified and characterized the biological function of the autophagy-related protein Atg6 in B. cinerea. Targeted deletion of the BcATG6 gene showed block of autophagy and several phenotypic defects in aspects of mycelial growth, conidiation, sclerotial formation and virulence. All of the phenotypic defects were restored by targeted gene complementation. Taken together, these results suggest that BcAtg6 plays important roles in the regulation of various cellular processes in B. cinerea.

Desferrioxamine B-Mediated Pre-Clinical In Vivo Imaging of Infection by the Mold Fungus Aspergillus fumigatus

Fungal infections are a serious threat, especially for immunocompromised patients. Early and reliable diagnosis is crucial to treat such infections. The bacterially produced siderophore desferrioxamine B (DFO-B) is utilized by a variety of microorganisms for iron acquisition, while mammalian cells lack the uptake of DFO-B chelates. DFO-B is clinically approved for a variety of long-term chelation therapies. Recently, DFO-B-complexed gallium-68 ([68Ga]Ga-DFO-B) was shown to enable molecular imaging of bacterial infections by positron emission tomography (PET). Here, we demonstrate that [68Ga]Ga-DFO-B can also be used for the preclinical molecular imaging of pulmonary infection caused by the fungal pathogen Aspergillus fumigatus in a rat aspergillosis model. Moreover, by combining in vitro uptake studies and the chemical modification of DFO-B, we show that the cellular transport efficacy of ferrioxamine-type siderophores is impacted by the charge of the molecule and, consequently, the environmental pH. The chemical derivatization has potential implications for its diagnostic use and characterizes transport features of ferrioxamine-type siderophores.

First report of Sclerotinia sclerotiorum on watercress (Nasturtium officinale) in aquaponic system in Hungary

Watercress (Nasturtium officinale) is an aquatic dicotyledonous vegetable belonging to Brassicaceae (Aiton 1812). Watercress was grown in an aquaponic system on fired clay ball medium at the Aquaponic Research Station of the University of Debrecen, in the city of Debrecen (Hungary). During January 2020, 3-month-old plants showed symptoms in aquaponic cultivation. A visual survey showed 30% of plants with symptoms. Leaves and stems withered and showed white cotton-like mycelium. Mycelia from infected plants were placed on potato dextrose agar (PDA) and incubated at 25°C for seven days. Single hyphal tips were transferred to produce a pure culture. All ten fungal isolates showed similar morphological characteristics on PDA. Colonies consisted of white mycelia after three days and globoid to irregular and black 2.5 to 7 (average, 3) mm (n = 100 from ten plates) sclerotia formed ten days later, which are the typical morphological features of Sclerotinia sclerotiorum (Mordue et al. 1976). Molecular identification was performed with one of the ten isolates (Scl_B). Mycelia were grown in 250 ml of potato dextrose broth in a rotary shaker at 175 rpm at 24°C for six days. DNA was extracted from mycelium using a Nucleospin plant II (Macherey-Nagel, Germany) according to the manufacturer’s protocol. PCR amplification (Kim et al. 2014) was performed with primers ITS1/ITS4 for the internal transcribed spacer region (White et al. 1990) on a Primus 96 thermal cycler (MWG Biotech, Germany). Specific polymerase chain reaction was performed with primers SSasprF/SSasprR (Abd-Elmagid et al. 2013). PCR products were sequenced by Microsynth Austria GmbH. NCBI BLAST analysis of the 440-bp ITS sequence (Genbank MW012403.1) showed 100% identity with the sequence of S. sclerotiorum (MT177267.1, etc.). The 170-bp specific gene sequence (Genbank MW959042.1) had a 100% similarity to hypothetical proteins (Genbank MK028159.1), with a 99.4% similarity to a portion of the S. sclerotiorum aspartyl protease gene (AF271387.1). Pathogenicity tests were carried out by inoculating surface-disinfested, 30-day-old watercress plants in plastic pots (15x15x12 cm). In three repeated experiments 90 watercress plants were measured. 15 plants (one plant per pot) were planted into the five-times autoclaved substrate (Biorgmix: pH 6.1±0.5%, N:1.5%, P2O5:0.7%, K2O:0.5%, organic matter content:50%) and inoculated by ten wheat kernels that were colonized by S. sclerotiorum (Scl_B) (Garibaldi et al. 2019). 15 plants were planted into the substrate with ten non-inoculated kernels as a control. Plants were kept in an MLR-352 climatic test chamber (PHCbi, Japan) at 21 ± 1°C for 12 hr light:dark cycle. On the first day of the experiment complex nutrient solution (Tek-Land: N:5%, P2O5:5%, K2O:5%, B:0.01%, Cu:0,01%, Mn:0.02%, Mo:0.002%, Zn:0.016%) was used, then autoclaved water daily. Eight days later white mycelium appeared on every inoculated plant and five days later dark sclerotia formed on the stems. Based on the morphological characteristics the re-isolated pathogen was S. sclerotiorum. Similar results were detected in three repeated experiments with white mold fungus being reisolated from all 45 infected watercress plants. The 45 non-inoculated plants did not show any symptoms and any diseases. This pathogen has already been reported on watercress in the field (Farr et al. 1989; Boland and Hall 1994; Garibaldi et al. 2019). This is the first reported case of white mold on watercress in aquaponic system in Hungary.

SORPTION OF HEAVY METALS BY MOLD FUNGUS ASPERGILLUS CARBONARIUS MELANIZING CARDBOARD

It has been shown that the mould fungus Aspergillus carbonarius, which synthesizes extracellular melanin, is able to develop due to the degradation of cellulose inside the cardboard under conditions of low water availability. The maximum yield of melanin was noted in a slightly alkaline medium, but in the presence of copper ions, a high level of pigmentation of the medium is also observed at low pH values. Melanized mycelium and exomelanin are characterized by a high sorption capacity in relation to heavy metal ions present in printing pigments of waste paper. In the process of growth A. carbonarius decreases acidity from neutral values to pH 2.8–3.1, increases the mobility of heavy metals immobilized on cellulose fibers and binds them by functional groups via ionic or chelating pathways. The sorption capacity of biomass with respect to copper, zinc, and nickel ions increased in the order of viable mycelium < inactivated mycelium < exomelanin. Lead ions were most actively bound by inactivated mycelium. The extracellular pigment accumulated copper better than other metals. The distribution coefficient in the system melanin – Cu2+ reached 1390 ml/g.

Egg masses as training aids for spotted lanternfly Lycorma delicatula detection dogs

The spotted lanternfly (Lycorma delicatula) is an invasive species first detected in 2014. The insect feeds on plants causing severe damage in vineyards such as the occurrence of sooty mold fungus that impairs leaf photosynthesis. Currently, there is extensive research on how to track and ultimately prevent the spread of this species. It lays eggs that persist through the winter, while the adults die out, which presents a unique opportunity to enter infested or suspected infested areas to begin quarantine and management of the spread while the species is dormant. Detection dogs may be a tool that can be used to search out the spotted lanternfly egg masses during this overwintering period, however it is not known whether dogs can detect any specific odor from the spotted lanternfly eggs. Moreover, as the eggs are only available during certain times of the year, and hatch based on temperature, finding training aids for the dogs could prove difficult. In this study, we investigated whether three detection dogs could learn the odor from dead spotted lanternfly egg masses and if so, whether that would allow them to recognize live spotted lanternfly egg masses. We found that dogs could be trained to find dead spotted lanternfly egg masses, and could learn to ignore relevant controls, with high levels of sensitivity and specificity (up to 94.6% and 92.8%, respectively). Further, we found that after the training, dogs could find live spotted lanternfly egg masses without additional training and returned to previous levels of sensitivity and specificity within a few sessions. Coded videos of training and testing sessions showed that dogs spent more time at the egg masses than at controls, as expected from training. These results suggest that dead spotted lanternfly egg masses could be a useful training aid for spotted lanternfly detection dogs.

Characterization of microbial antifreeze protein with intermediate activity suggests that a bound-water network is essential for hyperactivity

Antifreeze proteins (AFPs) inhibit ice growth by adsorbing onto specific ice planes. Microbial AFPs show diverse antifreeze activity and ice plane specificity, while sharing a common molecular scaffold. To probe the molecular mechanisms responsible for AFP activity, we here characterized the antifreeze activity and crystal structure of TisAFP7 from the snow mold fungus Typhula ishikariensis. TisAFP7 exhibited intermediate activity, with the ability to bind the basal plane, compared with a hyperactive isoform TisAFP8 and a moderately active isoform TisAFP6. Analysis of the TisAFP7 crystal structure revealed a bound-water network arranged in a zigzag pattern on the surface of the protein’s ice-binding site (IBS). While the three AFP isoforms shared the water network pattern, the network on TisAFP7 IBS was not extensive, which was likely related to its intermediate activity. Analysis of the TisAFP7 crystal structure also revealed the presence of additional water molecules that form a ring-like network surrounding the hydrophobic side chain of a crucial IBS phenylalanine, which might be responsible for the increased adsorption of AFP molecule onto the basal plane. Based on these observations, we propose that the extended water network and hydrophobic hydration at IBS together determine the TisAFP activity.

Heat Shock Protein HSP24 Is Involved in the BABA-Induced Resistance to Fungal Pathogen in Postharvest Grapes Underlying an NPR1-Dependent Manner

Although heat shock proteins (HSPs), a family of ubiquitous molecular chaperones, are well characterized in heat stress-related responses, their function in plant defense remains largely unclear. Here, we report the role of VvHSP24, a class B HSP from Vitis vinifera, in β-aminobutyric acid (BABA)-induced priming defense against the necrotrophic fungus Botrytis cinerea in grapes. Grapes treated with 10 mmol L–1 BABA exhibited transiently increased transcript levels of VvNPR1 and several SA-inducible genes, including PR1, PR2, and PR5. Additionally, phytoalexins accumulated upon inoculation with the gray mold fungus B. cinerea, which coincided with the action of a priming mode implicated in pathogen-driven resistance. Intriguingly, electrophoretic mobility shift (EMSA), yeast two-hybrid (Y2H) and His pull-down assays demonstrated that the nuclear chaperone VvHSP24 cannot modulate the transcript of PR genes but does directly interact with VvNPR1 in vivo or in vitro. Furthermore, we found that VvHSP24 overexpression enhanced the transcript levels of NPR1 and SA-responsive genes (PR1, PR2, and PR5) and increased the resistance of transgenic Arabidopsis thaliana to B. cinerea compared with wildtype Col-0. An opposite trend between CRISPR mutants of AtHSFB1 (the orthologous gene of VvHSP24 in Arabidopsis) and wildtype plants was observed. Hence, our results suggest that VvHSP24 has a potential role in NPR1-dependent plant resistance to fungal pathogen. BABA-induced priming defense in grapes may require posttranslational modification of the chaperone VvHSP24 to activate VvNPR1 transcript, leading to PR gene expressions and resistance phenotypes.

Cyclophilin BcCyp2 Regulates Infection-Related Development to Facilitate Virulence of the Gray Mold Fungus Botrytis cinerea

Cyclophilin (Cyp) and Ca2+/calcineurin proteins are cellular components related to fungal morphogenesis and virulence; however, their roles in mediating the pathogenesis of Botrytis cinerea, the causative agent of gray mold on over 1000 plant species, remain largely unexplored. Here, we show that disruption of cyclophilin gene BcCYP2 did not impair the pathogen mycelial growth, osmotic and oxidative stress adaptation as well as cell wall integrity, but delayed conidial germination and germling development, altered conidial and sclerotial morphology, reduced infection cushion (IC) formation, sclerotial production and virulence. Exogenous cyclic adenosine monophosphate (cAMP) rescued the deficiency of IC formation of the ∆Bccyp2 mutants, and exogenous cyclosporine A (CsA), an inhibitor targeting cyclophilins, altered hyphal morphology and prevented host-cell penetration in the BcCYP2 harboring strains. Moreover, calcineurin-dependent (CND) genes are differentially expressed in strains losing BcCYP2 in the presence of CsA, suggesting that BcCyp2 functions in the upstream of cAMP- and Ca2+/calcineurin-dependent signaling pathways. Interestingly, during IC formation, expression of BcCYP2 is downregulated in a mutant losing BcJAR1, a gene encoding histone 3 lysine 4 (H3K4) demethylase that regulates fungal development and pathogenesis, in B. cinerea, implying that BcCyp2 functions under the control of BcJar1. Collectively, our findings provide new insights into cyclophilins mediating the pathogenesis of B. cinerea and potential targets for drug intervention for fungal diseases.