Fungal Conidia Research Articles

Powdery Mildew of Echinacea purpurea Caused by Podosphaera xanthii in China.

Echinacea purpurea (Eastern Purple Coneflower) is a perennial herbaceous plant belonging to the Asteraceae. It originated from North America and is cultivated all over the world. Extracts of E. purpurea are widely used for the treatments of colds and sore throats (Jiao et al. 2020). In October 2024, powdery mildew-like signs and symptoms were observed on leaves of E. purpurea plants (n=100) cultivated in a garden (20 m2) in Xinxiang city, Henan Province, China (35.32° N, 113.92° E). A specimen (PX-ZM20241024) was stored in Xinxiang Key Laboratory of Plant Stress Biology. The infected leaves were covered with white and thin masses and showed senescence symptoms. More than 80% of plants (n=100) exhibited these signs and symptoms. White colonies were on both sides surfaces of the leaves, covering about 80% of the leaf area. The slightly curved or straight Conidia chain (n = 50) were 70 to 156 × 8 to 12 μm in size and consisted of foot cells, shorter cells, and conidia. Foot cells were straight, 30 to 60 μm long. Conidia were ellipsoid to oval, 20 to 35 × 12 to 17 μm (n = 50), with a length/width ratio of 1.8 to 2.2, containing fibrosin bodies. These morphological characteristics were similar to Podosphaera xanthii (Braun and Cook 2012). Following a previously reported method (Zhu et al. 2022), the internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions of a selected specimen of the fungus were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990) and GAPDH1/GAPDH3R (Bradshaw et al. 2022). The resulting sequences were deposited in GenBank (Accession No. PQ508348 and PQ734986). BLASTn analysis indicated that the ITS and GAPDH sequences had 99.82 % (564/565 bp) and 100% (238/238 bp) identity with reported P. xanthii (MW300956 and ON075660) on Xanthium strumarium and Cucurbita moschata, respectively (Zhu et al. 2024; Bradshaw et al. 2022). By phylogenetic analysis, the isolated fungus clustered with previously reported P. xanthii (Zhu et al. 2024; Bradshaw et al. 2022). Therefore, the morphology and phylogenetic analysis indicated that the pathogen was P. xanthii. To complete Koch's postulates, mature leaves of three healthy E. purpurea (30 cm high) were inoculated with fungal conidia by gently pressing surfaces of infested leaves onto leaves of healthy plants. Three untreated plants served as controls. Both the control and inoculated plants were separately placed in greenhouses (humidity, 60%; light/dark, 16 h/8 h; temperature, 18°C). 10 to 12 days post inoculation, the leaves of the inoculated plants exhibited signs of powdery mildew, whereas the control group remained unaffected. The experiments were repeated three times and the same results were obtained. Therefore, the pathogenicity of this fungal pathogen was confirmed. Previously, P. xanthii was reported on E. purpurea in Korea (Choi et al. 2020). To the best of our knowledge, this is the first report of P. xanthii on E. purpurea in China. The sudden presence of powdery mildew caused by P. xanthii may adversely affect plant health and thus reduce the medicinal value of E. purpurea. Therefore, the identification and confirmation of P. xanthii infecting E. purpurea enhances our comprehension of hosts of this pathogen and provides fundamental information for forthcoming disease control studies.

Mechanisms of regulated cell death during plant infection by the rice blast fungus Magnaporthe oryzae.

Fungi are the most important group of plant pathogens, responsible for many of the world's most devastating crop diseases. One of the reasons they are such successful pathogens is because several fungi have evolved the capacity to breach the tough outer cuticle of plants using specialized infection structures called appressoria. This is exemplified by the filamentous ascomycete fungus Magnaporthe oryzae, causal agent of rice blast, one of the most serious diseases affecting rice cultivation globally. M. oryzae develops a pressurized dome-shaped appressorium that uses mechanical force to rupture the rice leaf cuticle. Appressoria form in response to the hydrophobic leaf surface, which requires the Pmk1 MAP kinase signalling pathway, coupled to a series of cell-cycle checkpoints that are necessary for regulated cell death of the fungal conidium and development of a functionally competent appressorium. Conidial cell death requires autophagy, which occurs within each cell of the spore, and is regulated by components of the cargo-independent autophagy pathway. This results in trafficking of the contents of all three cells to the incipient appressorium, which develops enormous turgor of up to 8.0 MPa, due to glycerol accumulation, and differentiates a thickened, melanin-lined cell wall. The appressorium then re-polarizes, re-orienting the actin and microtubule cytoskeleton to enable development of a penetration peg in a perpendicular orientation, that ruptures the leaf surface using mechanical force. Re-polarization requires septin GTPases which form a ring structure at the base of the appressorium, which delineates the point of plant infection, and acts as a scaffold for actin re-localization, enhances cortical rigidity, and forms a lateral diffusion barrier to focus polarity determinants that regulate penetration peg formation. Here we review the mechanism of regulated cell death in M. oryzae, which requires autophagy but may also involve ferroptosis. We critically evaluate the role of regulated cell death in appressorium morphogenesis and examine how it is initiated and regulated, both temporally and spatially, during plant infection. We then use this synopsis to present a testable model for control of regulated cell death during appressorium-dependent plant infection by the blast fungus.

Powdery Mildew of Xanthium strumarium Caused by Podosphaera xanthii in central China.

Xanthium strumarium, known as cocklebur, is an annual herb and has been used in traditional Chinese medicine. In October 2020, powdery mildew-like disease signs and symptoms were observed on X. strumarium grown in a crop field, Xinxiang city, Henan Province, China (35.36076° N, 113.93467° E). The specimen (PX-XS2023) was stored in Xinxiang Key Laboratory of Plant Stress Biology. White colonies in irregular or coalesced circular shaped-lesions were abundant on both ad- and abaxial surfaces of leaves and covered up to 99 % of the leaf area. Some of the infected leaves were senesced. More than 70 % of plants (n = 130) exhibited these signs and symptoms. Conidiophores were straight or slightly curved, 55 to 160 × 11 to 13 μm composed of foot-cells, shorter cells and conidia. Conidia were ellipsoid to oval, 29 to 40 × 14 to 20 μm (n = 50), with a length/width ration of 2.0 to 2.5, containing fibrosin bodies. Dark brown to black chasmothecia were found on infected leaves. The appendages were mycelium-shaped and at the base of scattered or gregarious chasmothecia (n = 50, 70 to 120 μm in diameter). Asci were 55 to 80 × 50 to 65 μm (n=30). These morphological characteristics were consistent with those of Podosphaera xanthii (Braun and Cook 2012). The internal transcribed spacer (ITS) region and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) region of the fungus (PX-XS2023) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990) and GAPDH1/GAPDH3R (Bradshaw et al. 2022) according to a previously reported method (Zhu et al. 2022). The resulting sequences were respectively deposited into GenBank (Accession No. MW300956 and PP236083). BLASTn analysis indicated that the sequences were respectively 99.82 % (564/565) and 100% (272/272) identical to P. xanthii (MT260063 and ON075658). The phylogenetic analysis indicated that the strain PX-XS2023 and P. xanthii were clustered into a same branch. Therefore, the causal agent of powdery mildew on X. strumarium was P. xanthii. To conduct pathogenicity assays, mature leaves of five healthy X. strumarium (height in 50 centimeters) were inoculated with fungal conidia by gently pressing surfaces of infested leaves onto leaves of healthy plants (Zhu et al. 2020). Five untreated plants served as controls. The controls and inoculated plants were separately maintained in greenhouses (humidity, 60%; light/dark, 16 h/8 h; temperature, 18°C). Eight days post-inoculation, signs of powdery mildew were detectable on inoculated plants, however, the controls were asymptomatic. Thus, the fungal pathogen was morphologically and molecularly identified and confirmed as P. xanthii. This powdery mildew caused by P. xanthii was previously reported on X. strumarium in Korea, Russia and India (Farr and Rossman, 2021). In addition, P. xanthii was recorded on X. strumarium in Xinjiang Province, China (Tai 1979). However, this is the first report of P. xanthii on X. strumarium in central China, where is around 3000 km away from Xinjiang Province with geographically differences. The sudden presence of powdery mildew caused by P. xanthii may adversely affect plant health and thus reduce medical value of X. strumarium. Therefore, the identification and confirmation of P. xanthii infecting X. strumarium enhance the knowledge on the hosts of this pathogen in China and will provide fundamental information for disease control in the future.

First report of root rot of goji (Lycium barbarum), caused by Fusarium sambucinum

In July 2022, root rot was observed in several goji (Lycium barbarum) orchards located in Qinghai Province, China. Approximately 40% of the goji plants were affected in the orchards. Morphology of fungi isolated from affected plant, phylogenetic analyses, using internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF), and trichothecene (Tri5) sequences, as well as pathogenicity assays, were conducted to characterize and identify the causing agent of goji root rot. Isolate GQGF1-3 caused typical symptoms of L. barbarum root rot. Fungal colony characteristics and conidium morphology, combined with ITS, TEF, and Tri5 sequences showed that isolate GQGF1-3 was Fusarium sambucinum. This is the first report of F. sambucinum causing root rot of goji.

Minor impact of probiotic bacteria and egg white on Tenebrio molitor growth, microbial composition, and pathogen infection.

The industrial rearing of the yellow mealworm (Tenebrio molitor) for feed and food purposes on agricultural by-products may expose larvae and adults to entomopathogens used as biocontrol agents in crop production. Bacterial spores/toxins or fungal conidia from species such as Bacillus thuringiensis or Metarhizium brunneum could affect the survival and growth of insects. Therefore, the aim of this study was to investigate the potential benefits of a wheat bran diet supplemented with probiotic bacteria and dried egg white on larval development and survival and its effects on the gut microbiome composition. Two probiotic bacterial species, Pediococcus pentosaceus KVL B19-01 and Lactiplantibacillus plantarum WJB, were added to wheat bran feed with and without dried egg white, as an additional protein source, directly from neonate larval hatching until reaching a body mass of 20 mg. Subsequently, larvae from the various diets were exposed for 72 h to B. thuringiensis, M. brunneum, or their combination. Larval survival and growth were recorded for 14 days, and the bacterial microbiota composition was analyzed using 16S rDNA sequencing prior to pathogen exposure and on days 3 and 11 after inoculation with the pathogens. The results showed increased survival for T. molitor larvae reared on feed supplemented with P. pentosaceus in the case of co-infection. Larval growth was also impacted in the co-infection treatment. No significant impact of egg white or of P. pentosaceus on larval growth was recorded, while the addition of Lb. plantarum resulted in a minor increase in individual mass gain compared with infected larvae without the latter probiotic. On day 14, B. thuringiensis was no longer detected and the overall bacterial community composition of the larvae was similar in all treatments. On the other hand, the relative operational taxonomic unit (OTU) abundance was dependent on day, diet, and probiotic. Interestingly, P. pentosaceus was present throughout the experiments, while Lb. plantarum was not found at a detectable level, although its transient presence slightly improved larval performance. Overall, this study confirms the potential benefits of some probiotics during the development of T. molitor while underlining the complexity of the relationship between the host and its microbiome.

Plant Growth Promotion and Plant Disease Suppression Induced by Bacillus amyloliquefaciens Strain GD4a.

Botrytis cinerea, the causative agent of gray mold disease (GMD), invades plants to obtain nutrients and disseminates through airborne conidia in nature. Bacillus amyloliquefaciens strain GD4a, a beneficial bacterium isolated from switchgrass, shows great potential in managing GMD in plants. However, the precise mechanism by which GD4a confers benefits to plants remains elusive. In this study, an A. thaliana-B. cinerea-B. amyloliquefaciens multiple-scale interaction model was used to explore how beneficial bacteria play essential roles in plant growth promotion, plant pathogen suppression, and plant immunity boosting. Arabidopsis Col-0 wild-type plants served as the testing ground to assess GD4a's efficacy. Additionally, bacterial enzyme activity and targeted metabolite tests were conducted to validate GD4a's potential for enhancing plant growth and suppressing plant pathogens and diseases. GD4a was subjected to co-incubation with various bacterial, fungal, and oomycete pathogens to evaluate its antagonistic effectiveness in vitro. In vivo pathogen inoculation assays were also carried out to investigate GD4a's role in regulating host plant immunity. Bacterial extracellular exudate (BEE) was extracted, purified, and subjected to untargeted metabolomics analysis. Benzocaine (BEN) from the untargeted metabolomics analysis was selected for further study of its function and related mechanisms in enhancing plant immunity through plant mutant analysis and qRT-PCR analysis. Finally, a comprehensive model was formulated to summarize the potential benefits of applying GD4a in agricultural systems. Our study demonstrates the efficacy of GD4a, isolated from switchgrass, in enhancing plant growth, suppressing plant pathogens and diseases, and bolstering host plant immunity. Importantly, GD4a produces a functional bacterial extracellular exudate (BEE) that significantly disrupts the pathogenicity of B. cinerea by inhibiting fungal conidium germination and hypha formation. Additionally, our study identifies benzocaine (BEN) as a novel small molecule that triggers basal defense, ISR, and SAR responses in Arabidopsis plants. Bacillus amyloliquefaciens strain GD4a can effectively promote plant growth, suppress plant disease, and boost plant immunity through functional BEE production and diverse gene expression.

First Report of Nigrospora hainanensis Causing Leaf Blight on Brachiaria Griseb in China.

Brachiaria Griseb is an important gramineous forage grown in tropical regions, and also a main grass species uses to restore grasslands in tropical and subtropical regions of China. In August 2022, symptoms of leaf blight were observed on nearly 30% of the Brachiaria forage grass in the base of the Chinese Academy of Tropical Agricultural Sciences, Hainan, China. Symptomatic leaves initially exhibited small, reddish-brown, round or oval spots on their tips, subsequently expanding in size along the leaf margin, and gradually becoming wilted and dry. Twenty leaves showing typical symptoms were randomly collected and pieces (5×5 mm) from the junction of diseased and healthy region were cut, sterilized with 75% alcohol for 30 s, followed by 5% sodium hypochlorite for 30 s. Rinsed three times with sterile water and dried with sterile filter paper. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 28℃. The colonies were white on the surface and gray on the reverse side. The conidiogenous cells were monoblastic, hyaline, globose or ampulliform, and 6 to 8.7(13.1) ×5 to 7.2 (9) m (n=200). Conidia is single celled, smooth, black, spherical, or ellipsoidal, and (11)13 to 16.5 × (8.2) 10.3 to16.1 m (n=100). Setae were not observed. The morphological characteristics of the isolates were consistent with Nigrospora species. A representative isolate (LNH-5) was selected for genomic DNA extraction. Sequences of the transcribed spacer region of rDNA (ITS), partial translation elongation factor (TEF1), and beta-tubulin fragment (TUB) were amplified using primer pairs ITS1/ITS4(White et al. 1990), EF-728F and EF-986R (Carbone et al. 1999) and Bt2a and Bt2b (Glass et al. 1995), respectively. The sequences of ITS (OQ473493), TEF1 (OQ506059) and TUB gene (OQ506055) were submitted to GenBank. They were 99 to 100% identical to the Nigrospora hainanensis ITS(OM283581.1)(538 out of 519 bp),TEF1(YK019415.1)(274 out of 276 bp),and TUB (OK086377.1)(405 out of 405 bp) sequences. The phylogenetic maximum likelihood analysis using the combined ITS, TEF1 and TUB sequences indicated that the isolate was part of the N. hainanensis clade (100% bootstrap value) that also contained the type isolate LC6979 of this species. Pathogenicity was tested on 15 healthy Brachiaria plants. Fungal conidia were harvested by flooding two-week-old single conidial cultures with sterile water, centrifuging, and adjusting the concentration to 107 spores/mL. Then 10 μL of conidial suspension was dropped onto the surfaces of leaves wounded with a sterile needle. Sterile distilled water was used for control treatment. The test was repeated three times. After inoculation, the plants were kept at 90~100% relative humidity at 25 to 28°C in a greenhouse for two weeks, and monitored daily for lesion development. Seven days post inoculation, all the inoculated leaves presented symptoms similar to those observed under natural conditions, while the control leaves showed no symptoms. The fungus was re-isolated from the diseased tissues by the single spore isolation method (Choi et al. 1999) to complete Koch's postulates. This pathogen has been reported on sugarcane in China (Raza et al., 2019; Zheng et al., 2022). To our knowledge, this is the first report of N. hainanensis causing leaf blight on Brachiaria plants in China.

The Plasma Membrane H+ ATPase CsPMA2 Regulates Lipid Droplet Formation, Appressorial Development and Virulence in Colletotrichum siamense.

Plasma membrane H+-ATPases (PMAs) play an important role in the pathogenicity of pathogenic fungi. Lipid droplets are important storage sites for neutral lipids in fungal conidia and hyphae and can be used by plant pathogenic fungi for infection. However, the relationship between plasma membrane H+-ATPase, lipid droplets and virulence remains unclear. Here, we characterized a plasma membrane H+-ATPase, CsPMA2, that plays a key role in lipid droplet formation, appresorial development and virulence in C. siamense. Deletion of CsPMA2 impaired C. siamense conidial size, conidial germination, appressorial development and virulence but did not affect hyphal growth. ΔCsPMA2 increased the sensitivity of C. siamense to phytic acid and oxalic acid. CsPMA2 was localized to lipids on the plasma membrane and intracellular membrane. Deletion of CsPMA2 significantly inhibited the accumulation of lipid droplets and significantly affected the contents of some species of lipids, including 12 species with decreased lipid contents and 3 species with increased lipid contents. Furthermore, low pH can inhibit CsPMA2 expression and lipid droplet accumulation. Overall, our data revealed that the plasma membrane H+-ATPase CsPMA2 is involved in the regulation of lipid droplet formation and affects appressorial development and virulence in C. siamense.

First Report of Alternaria alternata Causing Leaf Spot on Broccoli in Ecuador.

In Ecuador, broccoli (Brassica oleracea var. italica) production is located in the Andean region, specifically Cotopaxi-Ecuador (INEC, 2019). A leaf pathogen has been constantly observed in this area, showing brown circular necrosis surrounded by yellowish halo-like spots causing leaf death (Fig. 1a). This pathogen was believed to be Alternaria sp.; however, the species was not determined either using classical or molecular tools. In 2021, ten leaves showing similar symptoms were collected in Cotopaxi and sent for pathogen identification. Here, leaf explants (0.25 cm2) showing disease symptoms were surface sterilized with 2% sodium hypochlorite (NaClO) and 70% ethanol (C2H6O), rinsed with sterile water, and transferred to Potato Dextrose Agar (PDA) media. Petri dishes were incubated in darkness at 25°C for five days. The single hyphal tip method was used to purify the cultures on PDA. Fifteen pure isolates were obtained after incubating for 14 days. Isolates were incubated under blacklight for two days to induce fungal sporulation. All isolates presented early white cotton-like mycelium that later became dark green (Fig 1b). Under the microscope, we observed straight primary conidia in simple or branched chains. Also, the conidia were obclavate, long ellipsoids, moderate in size (19.5-43.9 μm in length, 7.1-17.2 μm in width), and septate with few longitudinal septa. Lastly, the conidium body can narrow itself into a secondary conidia (Fig 1c) (Woudenberg et al., 2013). According to colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al., 2013). Five isolates were randomly selected for DNA extraction and sequencing of ITS (internal transcribed spacer; Chou, H.H. and Wu, W.S. 2002), TEF (translation elongation factor; O'Donnell et al., 1998), and RPB2 (RNA polymerase II second largest subunit; Liu et al., 1999) gene regions. DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al., 2018). Consensus sequences were deposited in GenBank with the following accession numbers: ITS, ON982232; TEF, ON983964; RPB2, ON983963. A multilocus Bayesian inference phylogenetic tree was constructed in Beast software (version 1.8.4) using the concatenated sequences (Drummond et al., 2012; Maharachchikumbura et al., 2014). The isolates in our study clustered with isolates of Alternaria alternata, confirming their identity (Figure 2). For Koch's postulates, healthy broccoli plants were grown in sterile soil for six weeks. The fungal conidia were suspended in sterile distilled water (1×106 conidia/ml), and the leaves were inoculated by spraying the spore solution. The control treatment was sprayed with sterile distilled water alone. Plants were maintained at 28°C and had more than 85% relative humidity (Sigillo et al., 2020). Seven days after inoculation, plants showed chlorosis and necrosis. Ten days later, 100% of the treated leaves presented brown circular necrosis (Fig. 1d). Control plants showed no disease symptoms. Re-isolation of the pathogen from the diseased leaf tissue was performed as previously described. The isolates presented the exact morphology of pure cultures obtained from field-diseased leaves. The pathogenicity test was performed twice. To our knowledge, this is the first report on A. alternata being the causal agent of leaf spot on broccoli in Ecuador. Disease diagnosis contributes to providing strategies against this pathogen. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in broccoli.

Screening of Fungal Strains and Formulations of Metarhizium anisopliae to Control Phyllotreta striolata in Chinese Flowering Cabbage.

(1) Background: The cabbage flea beetle (CFB; Phyllotreta striolata) seriously damages the production of Chinese flowering cabbage (CFC; Brassica campestris L. ssp. chinensis var. utilis), which is a key leafy vegetable in South China. A large number of chemical insecticides have been sprayed to control this pest; as a result, residues and resistances are becoming an issue. It is necessary to develop biocontrol technologies to address this issue. (2) Methods: Fungal strains were selected based on bioactivity against CFB, and CFC seed pelletization with fungal conidia was subject to evaluation of control efficacy against CFB. The effective mixture of fungus and chemical insecticide was determined based on safety and joint toxicology tests. (3) Results: The screening of 103 strains from 14 genera identified the Metarhizium anisopliae strain MaGX19S02 (Ma) as the one with the highest virulence. The LC50s of Ma to CFB adult and second instar larvae on day 9 post-treatment were 3.04 × 106 and 27.2 × 106 spores/mL, respectively. In the pot test, the pelletization of CFC seeds with Ma conidia (50/25/12.5 mg in 1 g seed with 4 g fillers) demonstrated significant CFB mortalities (45-82%) 20 days after the larvae were introduced. In the field test, the seed pelletization achieved 57-81% control efficacy 14 days after sowing. Furthermore, the combination of Ma with chlorfenapyr (Chl) demonstrated a synergistic effect against CFB; based on this result, we prepared the mixture formulation of 20% Ma-Chl wettable powder (WP). The assessment of the effects of 20% Ma-Chl WP (500× diluent) against CFB revealed 93.33% mortality in the pot test and 61.3% control efficacy in the field test on day 7 post-treatment. (4) Conclusions: The findings demonstrate the potential of Ma to control CFB in the field. Seed pelletization with Ma conidia effectively controlled CFB larvae and protected CFC seedlings, wherein a mixture formulation of 20% Ma-Chl WP had substantial efficacy in controlling CFB adults. Our research provides new methods for CFB biocontrol.

Neopestalotiopsis sp. and Colletotrichum karstii, causal agents of leaf spots on camedor palm (Chamaedorea quezalteca) in Mexico

Since 2020, camedor palm (Chamaedorea quezalteca) plantations in the Frailesca region, Chiapas, Mexico, have been affected by foliar diseases that induce leaf spot and anthracnose symptoms that reduce production, leaf quality and thus the loss of their commercial value. The objectives of this work were to identify the causal agents and to quantify the incidence and severity of foliar diseases in plantations of camedor palm. Two fungi were recurrently isolated, one from leaves with leaf spot symptoms and the other with anthracnose, both were identified morphologically and molecularly by sequencing the internal transcribed spacer (ITS5/ITS4), pathogenicity tests were performed on healthy plants where fungal conidia were inoculated, and in the field the incidence and severity of the disease were quantified with the support of a pictographic scale. The fungi identified were Neopestalotiopsis sp. in leaf spot symptoms and Colletotricum karstii in anthracnose, both pathogens reproduced the same symptoms observed in the field, by means of pathogenicity tests. Disease incidence and severity percentages were transformed to area under the disease progress curve (AUDPC), these diseases presented an annual average incidence and severity of 35.33% (AUDPC= 14180) and 39.16% (AUDPC=12885) for leaf spot, 19.5% (AUDPC= 7123) and 55.83% (AUDPC= 20380) for anthracnose, respectively.

Surrogate infection model predicts optimal alveolar macrophage number for clearance of Aspergillus fumigatus infections

The immune system has to fight off hundreds of microbial invaders every day, such as the human-pathogenic fungus Aspergillus fumigatus. The fungal conidia can reach the lower respiratory tract, swell and form hyphae within six hours causing life-threatening invasive aspergillosis. Invading pathogens are continuously recognized and eliminated by alveolar macrophages (AM). Their number plays an essential role, but remains controversial with measurements varying by a factor greater than ten for the human lung. We here investigate the impact of the AM number on the clearance of A. fumigatus conidia in humans and mice using analytical and numerical modeling approaches. A three-dimensional to-scale hybrid agent-based model (hABM) of the human and murine alveolus allowed us to simulate millions of virtual infection scenarios, and to gain quantitative insights into the infection dynamics for varying AM numbers and infection doses. Since hABM simulations are computationally expensive, we derived and trained an analytical surrogate infection model on the large dataset of numerical simulations. This enables reducing the number of hABM simulations while still providing (i) accurate and immediate predictions on infection progression, (ii) quantitative hypotheses on the infection dynamics under healthy and immunocompromised conditions, and (iii) optimal AM numbers for combating A. fumigatus infections in humans and mice.

Links between Soil Bacteriobiomes and Fungistasis toward Fungi Infecting the Colorado Potato Beetle.

Entomopathogenic fungi can be inhibited by different soil microorganisms, but the effect of a soil microbiota on fungal growth, survival, and infectivity toward insects is insufficiently understood. We investigated the level of fungistasis toward Metarhizium robertsii and Beauveria bassiana in soils of conventional potato fields and kitchen potato gardens. Agar diffusion methods, 16S rDNA metabarcoding, bacterial DNA quantification, and assays of Leptinotarsa decemlineata survival in soils inoculated with fungal conidia were used. Soils of kitchen gardens showed stronger fungistasis toward M. robertsii and B. bassiana and at the same time the highest density of the fungi compared to soils of conventional fields. The fungistasis level depended on the quantity of bacterial DNA and relative abundance of Bacillus, Streptomyces, and some Proteobacteria, whose abundance levels were the highest in kitchen garden soils. Cultivable isolates of bacilli exhibited antagonism to both fungi in vitro. Assays involving inoculation of nonsterile soils with B. bassiana conidia showed trends toward elevated mortality of L. decemlineata in highly fungistatic soils compared to low-fungistasis ones. Introduction of antagonistic bacilli into sterile soil did not significantly change infectivity of B. bassiana toward the insect. The results support the idea that entomopathogenic fungi can infect insects within a hypogean habitat despite high abundance and diversity of soil antagonistic bacteria.

A lipid droplet-associated protein Nem1 regulates appressorium function for infection of Magnaporthe oryzae

Lipid droplets are important storages in fungal conidia and can be used by plant pathogenic fungi for infection. However, the regulatory mechanism of lipid droplets formation and the utilization during fungal development and infection are largely unknown. Here, in Magnaporthe oryzae, we identified a lipid droplet-associated protein Nem1 that played a key role in lipid droplets biogenesis and utilization. Nem1 was highly expressed in conidia, but lowly expressed in appressoria, and its encoded protein was localized to lipid droplets. Deletion of NEM1 resulted in reduced numbers of lipid droplets and decreased content of diacylglycerol (DAG) or triacylglycerol (TAG). NEM1 was required for asexual development especially conidia production. The Δnem1 mutant was nearly loss of virulence to host plants due to defects in appressorial penetration and invasive growth. Remarkably, Nem1 was regulated by the TOR signaling pathway and involved in the autophagy process. The Ser303 residue of Nem1 could be phosphorylated by the cAMP-PKA signaling pathway and was important for biological function of Nem1. Together, our study revealed a regulatory mechanism of lipid biogenesis and metabolism during the conidium and appressorium formation of the rice blast fungus.

In Vitro Photoinactivation of Fusarium oxysporum Conidia with Light-Activated Ammonium Phthalocyanines.

Antimicrobial photodynamic therapy (aPDT) has been explored as an innovative therapeutic approach because it can be used to inactivate a variety of microbial forms (vegetative forms and spores) without causing significant damage to host tissues, and without the development of resistance to the photosensitization process. This study assesses the photodynamic antifungal/sporicidal activity of tetra- and octasubstituted phthalocyanine (Pc) dyes with ammonium groups. Tetra- and octasubstituted zinc(II) phthalocyanines (1 and 2) were prepared and tested as photosensitizers (PSs) on Fusarium oxysporum conidia. Photoinactivation (PDI) tests were conducted with photosensitizer (PS) concentrations of 20, 40, and 60 µM under white-light exposure at an irradiance of 135 mW·cm-2, applied during 30 and 60 min (light doses of 243 and 486 J·cm-2). High PDI efficiency corresponding to the inactivation process until the detection limit was observed for both PSs. The tetrasubstituted PS was the most effective, requiring the lowest concentration and the shortest irradiation time for the complete inactivation of conidia (40 µM, 30 min, 243 J·cm-2). Complete inactivation was also achieved with PS 2, but a longer irradiation time and a higher concentration (60 µM, 60 min, 486 J·cm-2) were necessary. Because of the low concentrations and moderate energy doses required to inactivate resistant biological forms such as fungal conidia, these phthalocyanines can be considered potent antifungal photodynamic drugs.

Entomopathogenic Fungi as a Potential Management Tool for the Control of Urban Malaria Vector, Anopheles stephensi (Diptera: Culicidae).

Anopheles stephensi (Diptera: Culicidae) is the vector of urban malaria in India and has a significant impact in transmitting infection in cities and towns. Further, WHO has also alarmed its invasive nature as a threat to African countries. Entomopathogenic fungi such as Beauveria bassiana and Metarhizium anisopliae have been found to be highly effective in controlling vector mosquito populations and therefore could be used in integrated vector control programs. Before employing the entomopathogenic fungi into the control programs, an effective isolate must be selected. Two separate experiments were conducted to evaluate the efficacy of Beauveria bassiana (Bb5a and Bb-NBAIR) and Metarhizium anisopliae (Ma4 and Ma-NBAIR) isolates against An. stephensi. Cement and mud panels were treated with fungal conidia with the concentration of 1 × 107 conidia/mL and adult An. stephensi mosquitoes were exposed to the treated panels (24 h after conidia were applied) by conducting WHO cone bioassay tests. The survival of the mosquitoes was monitored daily until the 10th day. In the second experiment, second instar larvae of An. stephensi were treated with fungal (Bb5a, Bb-NBAIR, Ma4 and Ma-NBAIR) conidia and blastospores with the spore concentration of 1 × 107 spores/mL. The survival of larvae was monitored until pupation. All the fungal isolates tested caused mortality in the adult mosquitoes, with varying median survival times. The Bb5a isolate reported lesser median survival times on both cement and mud panels (6 days). The treated mosquitoes showed similar survival rates for each fungal isolate irrespective of the panel type. There was no mortality in the treated larvae; however, a delay in larval development to pupae was observed compared with the untreated control larvae. Ma4-treated larvae took 11 days (95% CI = 10.7-11.2) to become pupae when compared with the untreated control larvae (6 days [95% CI = 5.6-6.3]). The findings of this study will be useful to consider EPF as a tool for the management of vector mosquitoes.

First report of Podosphaera fusca Causing Powdery Mildew on Coreopsis lanceolata in China.

Coreopsis lanceolata, known as lance-leaf coreopsis, is a perennial plant with high ornamental value. It is widely grown in many public parks and home gardens in China due to its showy flowers. From May to June 2020, typical powdery mildew-signs and symptoms were seen on leaves of C. lanceolata cultivated in the east campus of Henan Normal University, Henan Province, China. Abundant white powder-like masses in spot- or coalesced-lesions were on ad- and abaxial surfaces of plant leaves and covered up to 50 % of the leaf area. The infected leaves were deformed and eventually prematurely senescent. Approximately 80 % of observed C. lanceolata plants showed these signs and symptoms. Unbranched conidiophores (n = 25) were 90 to 200 × 12 to 20 μm and showed a foot cell, followed by 1 to 3 short cells and conidia. Ellipsoid-ovoid shaped conidia (n = 30) were 22 to 36 × 15 to 23 μm, with a length/width ratio of 1.4 to 2.4. No chasmothecia were detected. The powdery mildew fungus was initially identified as Podosphaera fusca based on the morphological characteristics. Total genomic DNA of the pathogen was extracted and the rDNA internal transcribed spacer (ITS) region was amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990; Zhu et al. 2019). The obtained sequence was deposited into GenBank under Accession No. MT899186 and was 100 % identical to P. fusca (JX546297) from Herba eupatorii (Ding et al. 2013). To perform pathogenicity assays, leaf surface of three healthy plants was inoculated with fungal conidia according to a previously described method (Zhu et al. 2021). As a control, three non-inoculated plants were used. The control and inoculated plants were placed separately in two growth chambers (light/dark, 16 h/8 h; humidity, 65 %; temperature, 20 ℃). Fourteen- to sixteen-days post inoculation, powdery mildew signs were noticed on inoculated plants, whereas control remained asymptomatic. Similar results were found by performing two repeated pathogenicity assays. Therefore, based on the morphological and molecular analysis, the pathogen was identified and confirmed as P. fusca. This fungus has been reported on C. lanceolata in Korea (Park et al. 2010) and Italy (Garibaldi et al. 2007). This is, to the best of our knowledge, the first report of P. fusca on C. lanceolata in China. The sudden occurrence of this powdery mildew disease on C. lanceolata may adversely affect the health of valuable ornamentals in China. The precise identification of the causal agent of this powdery mildew of C. lanceolata is a preliminary step in developing effective disease management strategies.

Characterization of Pseudogymnoascus destructans conidial adherence to extracellular matrix: Association with fungal secreted proteases and identification of candidate extracellular matrix binding proteins.

Pseudogymnoascus destructans is the etiological agent of white-nose syndrome (WNS), a fungal skin infection of hibernating bats. Pathophysiology of the disease involves disruption of bat metabolism and hibernation patterns, which subsequently causes premature emergence and mortality. However, information on the mechanism(s) and virulence factors of P. destructans infection is minimally known. Typically, fungal adherence to host cells and extracellular matrix (ECM) is the critical first step of the infection. It allows pathogenic fungi to establish colonization and provides an entry for invasion in host tissues. In this study, we characterized P. destructans conidial adherence to laminin and fibronectin. We found that P. destructans conidia adhered to laminin and fibronectin in a dose-dependent, time-dependent and saturable manner. We also observed changes in the gene expression of secreted proteases, in response to ECM exposure. However, the interaction between fungal conidia and ECM was not specific, nor was it facilitated by enzymatic activity of secreted proteases. We therefore further investigated other P. destructans proteins that recognized ECM and found glyceraldehyde-3-phosphate dehydrogenase and elongation factor 1-alpha among the candidate proteins. Our results demonstrate that P. destructans may use conidial surface proteins to recognize laminin and fibronectin and facilitate conidial adhesion to ECM. In addition, other non-specific interactions may contribute to the conidial adherence to ECM. However, the ECM binding protein candidates identified in this study highlight additional potential fungal virulence factors worth investigating in the P. destructans mechanism of infection in future studies.

Identification and characterization of Pestalotiopsis spp. causing leaf spot and leaf blight on jabon (Neolamarckia spp.) in Indonesia

Abstract. Herliyana EN, Oktavianto P, Siregar UJ. 2022. Identification and characterization of Pestalotiopsis spp. causing leaf spot and leaf blight on jabon (Neolamarckia spp.) in Indonesia. Biodiversitas 23: 6547-6556. Jabon (Neolamarckia spp.) is one of the fast-growing trees with high economic value for industrial plantations. Its monoculture planting system is faced with certain problems, such as the leaf spot and leaf blight diseases caused by Pestalotiopsis sp. Therefore, the aim of this research was to describe the morphology of Pestalotiopsis species through macroscopic and microscopic observations, followed by the identification of fungus using DNA barcoding causing leaf spot and leaf blight on the tree. A total of 11 Pestalotiopsis isolates were used for this research. The morphology was identified by observing mycelium colonies and fungal conidia, while DNA barcoding was initiated after DNA isolation using cetyltrimethylammonium bromide (CTAB) method, amplification with ITS 1 and 4 primers, followed by sequencing process. The results of DNA barcoding showed that 5 species of Pestalotiopsis, namely Pseudopestalotiopsis theae, Pestalotiopsis microspora, Pestalotiopsis palmarum, Neopestalotiopsis clavispora, and Pestalotiopsis virgatula were found associated with leaf spot and blight disease. All the five species were further classified into 3 clades using phylogeny according to their median cells and apical appendage shape. The clade X consisted of P. palmarum, N. clavispora, and P. virgatula with versicolorous median cells, whereas clade Y included P. microspora with concolorous median cell, and clade Z consisted Ps. theae with concolorous median cell and knobbed apical appendage.

Enhanced virulence of Beauveria bassiana against Thrips tabaci by the addition of bacterial metabolites derived from Xenorhabdus hominickii

• Immunosuppressive agents are supposed to enhance the virulence of microbial pathogens against insect pests. • The culture broth of Xenorhabdus hominickii inhibited immune responses of Thrips tabaci . • The addition of the bacterial broth to Beauveria bassiana enhanced the fungal insecticidal activity. Xenorhabdus and Photorhabdus are two bacterial genera specifically symbiotic to Steinernema and Heterorhabditis , which are the entomopathogenic nematode genera, respectively. These bacteria are well known to produce potent secondary metabolites suppressing insect immune responses. This study aimed to develop a potent microbial insecticide against the onion thrips, Thrips tabaci , using the bacterial metabolites. Among the chemical insecticides that have been used to control the thrips, spinosad was highly effective against both larvae and adults of T. tabaci . Three different entomopathogenic fungi were also effective to kill the thrips. However, the fungal virulence was much less than the control efficacy of the chemical insecticide, spinosad. To enhance the fungal virulence of Beauveria bassiana (Bb), the bacterial culture broth of Xenorhabdus / Photorhabdus was added to suppress the thrips immune defense. Among six different bacterial species, X. hominickii (Xh) produced highly potent metabolites to enhance the fungal virulence. Indeed, four different bacterial metabolites (GameXPeptide, benzylideneacetone, oxindole, and 3-ethoxy-4-methoxyphenol) of the bacteria suppressed the gene expressions of an antimicrobial peptide, lysozyme , which was highly inducible to the fungal infection. To optimize the mixture ratio of fungal and bacterial pathogens, the fungal conidia and bacterial culture broth were freeze-dried and mixed in different ratios. Laboratory and field assays showed that a mixture spray of freeze-dried Xh culture broth (3 g) and Bb conidia (1.17 × 10 9 conidia) in a liter was effective to control T. tabaci infesting welsh onion.