First Report of Coffee Canker Disease Caused by Ceratocystis fimbriata in China

Coffee (Coffea arabica L.) is one of the most important agricultural commodities in the world market. As an important cash crop in China, coffee is cultivated mainly in Yunnan and Hainan provinces. During October 2013 and September 2020, coffee trees showing typical dieback and wilt symptoms were found in Nanping town (N 22° 38', E 101° 0'), Pu’er, and Puwen town (N 22° 32', E 101° 4'), Xishuangbanna in Yunnan province, China. Symptomatic trees initially exhibited yellowing of foliage, expanding in size along the leaf margin, then became blighted and dry, and the internal xylem in main stem discolored brown to black. Infected trees eventually developed dieback and wilt. Disease incidence ranged from 10% to 22% and 25% to 40% of crown symptoms in the affected coffee trees. In extreme cases, 50% out of 380 trees were affected. The stems of coffee trees with typical symptoms were collected, and then the diseased tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, fungal mycelium was observed growing from the tissue. Three isolates (C3-1, C3-2, and C3-2-1) were obtained by picking spore masses from the apices of perithecia and transferring them to malt extract agar (MEA) medium and incubated at 25°C for 10 days to observe the cultural features. In culture, colonies reaching 65 mm within 10 days, mycelium initially white, then becoming light blue-green. After 6 days of formation, perithecia were black, globose (123.8 - 173.4 μm × 138.2 - 180.6 μm), and showed a long black neck (414.2 - 650.0 μm). Ascospores with outer cell wall forming a brim, hat-shaped, accumulating in a mucilaginous mass at the tips of ostiolar hyphae (4.3 μm × 6.0 μm). Cylindrical endoconidia (14.1 - 45.2 μm × 3.5 - 5.7 μm) were hyaline. Chain of barrel-shaped conidia (6.6 - 10.2 μm × 6.8 - 8.8 μm) were found. Aleuroconidia (10.8 - 16.9 μm × 9.1 - 13.0 μm) were olive-brown, ovoid or obpyriform, and smooth. Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata Ellis & Halst. (Engelbrecht and Harrington 2005). The three isolates were used for molecular identification, and their genomic DNA was extracted using the chelex-100 method (Xu et al. 2020). The internal transcribed spacer (ITS) region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of the ITS sequence data (GenBank accessions KY580836, KJ511480, and KJ511479) showed that the isolates were 100% homologous to isolates of C. fimbriata from Punica granatum, Camellia sinensis, and Cucumis sativus in China (GenBank accessions KY580891, KY580870, and MH535909, respectively) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis was performed using MEGA 6.06 based on the ITS sequences. The three isolates were clustered on the same clade with other C. fimbriata isolates with a high bootstrap value (90%). Therefore, the fungus was identified as C. fimbriata based on both morphological and molecular characteristics. Pathogenicity of the three isolates was tested by inoculating one-year-old pot grown coffee seedlings (C. arabica) through drenching the loams with 30 ml spore suspension (1 × 106 spores/ml). Control plants were inoculated with 30 ml of sterile distilled water. The trees were kept in a controlled greenhouse at 25°C and watered weekly. One month after inoculation, all inoculated plants produced typical dieback and wilt symptoms, whereas the control trees showed no symptoms. The same fungus was isolated from the inoculated trees on PDA and identified as C. fimbriata according to the methods described above, and no fungal growth was observed in the controls, thus fulfilling the Koch's postulates. Coffee canker disease caused by C. fimbriata has been reported in Indonesia and Colombia (Marin et al. 2003). To our knowledge, this is the first report of C. fimbriata causing canker disease of coffee trees in China.

First report of rubber tree wilt caused by Ceratocystis fimbriata in China

The rubber tree (Hevea brasiliensis) is an important economic resource for the rubber and latex industry. During November 2013 and June 2016, rubber trees showing typical wilt symptoms were found in Mengla, Xishuangbannan, Yunnan, China (N 21° 28', E 101° 33'). Symptomatic trees initially exhibited wilting of foliage on individual branches, then spread to the whole canopy, finally followed by death of the whole tree. Dark-blue to black discoloration was observed in the inner bark and affected xylem, a grayish layer of fungal growth and sporulation occasionally. The disease was detected on 20% of trees surveyed. The diseased tissues of three rubber trees were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA), and incubated at 25°C. After 7 days, a fungus was consistently observed growing from the tissue. Three single-spore isolates were obtained. In culture, colonies reaching 69 mm diam within 10 days, mycelium was initially white, then becoming celadon. After 5 days of perithecium formation, observed perithecia were black, globose (173.1 - 237.9 × 175.6 - 217.2 μm) and showed a long black neck (507.3 - 794.1 μm). Ascospore with outer cell wall forming a brim, hat-shaped at the tips of ostiolar hyphae (3.43 × 5.63 μm). Cylindrical endoconidia (10.5 - 39.7 × 3.5 - 6.6 μm) were hyaline. Chain of barrel-shaped conidia (7.2 - 9.5 × 4.1 - 6.2 μm) was found. Aleuroconidia were ovoid or obpyriform, and smooth (10.2 - 14.1 × 8.4 - 10.6 μm). Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata (Engelbrecht and Harrington 2005). The genomic DNA was extracted from isolates (XJm10-2-5, XJm8-2-5, XJm4) using the Chelex-100 method (Xu et al. 2020). The ITS region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of ITS sequence data (GenBank accessions KJ511488, KJ511485, KT963149) showed that the isolates were 100% homologous to those of the isolates on Punica granatum and Colocasia esculenta from China (GenBank accessions KT963152, MH793673) by BLAST analysis. Neighbor-joining phylogenetic analyse were performed using MEGA 6.06 based on ITS sequences (Fig. 1). Analyses showed that all isolates located on the same clade with all C. fimbriata with a high bootstrap support. Therefore, the fungus was identified as C. fimbriata based on morphology and molecular evidences. Pathogenicity of C. fimbriata isolated from this study was tested by inoculation of three one-year-old pot-grown (3L) seedlings of rubber tree. The soil of three seedlings was inoculated by drenching with 30 ml spore suspension (2.0 × 106 spores / ml). Three control plants were inoculated with 30 ml of sterile distilled water. The experiment was repeated three times. The plants were kept in a controlled greenhouse at 25°C and watered weekly. After the inoculation for one month, all the isolates produced typical wilt symptoms, while control plants showed no symptoms. The original fungus was successfully re-isolated from inoculated trees and identified as C. fimbriata according to the methods described above. The pathogenicity assay showed that C. fimbriata was pathogenic to rubber trees. C. fimbriata was first reported on rubber tree in Brazil (Albuquerque et al. 1972; Silveira et al. 1985). To the best of our knowledge, this is the first report of C. fimbriata causing wilt of rubber tree in China. This finding contributes to understanding the diversity of this pathogen, and it appears to be a significant threat to rubber trees in its ecosystem.

Single molecule dynamics of DNA receptor ComEA, membrane permease ComEC and taken up DNA in competent Bacillus subtilis cells

In competent Gram-negative and Gram-positive bacteria, double stranded DNA is taken up through the outer cell membrane and/or the cell wall, and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single stranded DNA, and transported through the cell membrane via ComEC. We show that in Bacillus subtilis , the C-terminus of ComEC, thought to act as a nuclease, is not only important for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single molecule tracking, we show that only 13% of ComEC molecules are statically localised at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C-terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as nuclease. By tracking fluorescently labeled DNA, we show that taken up DNA has a similar mobility as a protein, in spite of being a large polymer. DNA dynamics are similar within the periplasm as those of ComEA, suggesting that most taken up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken up DNA, before its entry into the cytosol. Importance Bacteria can take up DNA from the environment and incorporate it into their chromosome, termed “natural competence” that can result in the uptake of novel genetic information. We show that fluorescently labelled DNA moves within the periplasm of competent Bacillus subtilis cells, with similar dynamics as DNA receptor ComEA. This indicates that DNA can accumulate in the periplasm, likely bound by ComEA, and thus can be stored before uptake at the cell pole, via integral membrane DNA permease ComEC. Assembly of the latter assembles at the cell pole likely occurs by a diffusion-capture mechanism. DNA uptake into cells thus takes a detour through the entire periplasm, and involves a high degree of free diffusion along and within the cell membrane.

Candida albicans phosphate transport, facilitating nucleotide sugar biosynthesis, contributes to cell wall stability.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin signaling, oxidative stress resistance and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild type cells recovering from phosphate starvation. Non-phosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar,GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. Our model is that low substrate concentrations of beta-D-glucan- and chitin synthases diminish enzymatic reaction rates and potentiate pharmacologic inhibitors to decrease the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-D-glucans or chitin. Hence inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

Preservation of Features of Anatomical Polymorphism of Deschampsia antarctica É. Desv. (Poaceae) During In Vitro Clonal Reproduction

We studied the anatomical features of the leaf structure of Deschampsia antarctica É. Desv. (Poaceae) obtained from seeds collected from different habitats of the maritime Antarctic. These plants have been maintained in culture in vitro for more than 7 years. The plants include stable chromosome forms comprising diploids (2 n = 26), a diploid plant with B-chromosomes (2 n = 26 + 0–3B), a triploid with rearranged chromosome morphology, and myxoploids with different ratios of diploid and nondiploid cells in the root meristem. The D. antarctica plants that were studied generally had a similar anatomical structure in culture in vitro and in nature. At the same time, plants of different cultivated genotypes also displayed their own leaf structure features. In particular, qualitative features included asymmetric four-ribs and five-rib leaves instead of typical three-rib leaves for some genotypes, some individuals characterized by the presence of unicellular nonglandular pointed trichomes on an adaxial side, differences in vascular bundle sheath, and other features. No clear dependence of the anatomical structural features on the chromosomal status of the studied genotypes was evident. At the same time, differences were evident in traits that included the number of stomata, thickness of the outer cell walls of the epidermis, bundles sheath state, number of leaf ribs, and the persistent presence of trichomes under prolonged in vitro cloning. The findings indicate that D. antarctica plants collected from different locations of the maritime Antarctic for long-term cultivation in vitro under standard cultivation conditions retain the anatomical characteristics of the wild plants. The polymorphism of in vitro-cultivated plants is not related to the polymorphism of their karyotype, but is apparently due to the heterogeneity of the original plants and possibly the epigenetic fixation of a number of anatomical features produced in the natural microhabitats of D. antarctica populations.

Crosstalk between calcineurin and the cell wall integrity pathways prevents chitin overexpression in Candida albicans

Echinocandins such as caspofungin are front line antifungal drugs that compromise β-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by up-regulation of chitin synthesis, thereby sustaining cell wall integrity. When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a sub-population of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable super high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway.

1065. Efficacy of Cefiderocol in Experimental Stenotrophomonas maltophilia Pneumonia in Persistently Neutropenic Rabbits

Background Stenotrophomonas maltophilia causes lethal pneumonia, bacteremia, and sepsis in immunocompromised patients. As a standard of care, trimethoprim-sulfamethoxazole (T/S) is considered to be the first-line therapy for Stenotrophomonas pneumonia. Cefiderocol (CFDC) is a new parenteral siderophore cephalosporin that is transported through the outer cell membrane as a siderophore mimic that then inhibits Gram-negative cell wall biosynthesis. CFDC has potent activity in vitro against S. maltophilia; however, little is known about its in vivo activity against Stenotrophomonas pneumonia in immunocompromised hosts. We therefore studied CFDC in comparison to TS in the persistently neutropenic rabbit model of Stenotrophomonas pneumonia. This rabbit model, in contrast to conventional murine models, reflects the human pattern of infection more accurately over time. Methods We initially studied the plasma pharmacokinetics of CFDC in non-infected and infected animals. Stenotrophomonas pneumonia was established by direct endotracheal inoculation of S. maltophilia 1×1010 CFUs for tracheobronchial colonization that evolved into bronchopneumonia. Experimental groups consisted of CFDC, T/S, and untreated controls (UC). Rabbits received CFDC at 120 mg/kg IV Q8h and T/S at 5 mg/kg IV Q12h. Profound persistent neutropenia was maintained with cytosine arabinoside. Treatment was continued for 10 days. Results There were no significant differences between non-infected and infected rabbits in CFDC pharmacokinetics. Rabbits treated with CFDC and T/S demonstrated significant decreases of residual pulmonary and BAL bacterial burden vs UC (p≤0.001). CFDC achieved full clearance of S. maltophilia from lung tissue and BAL. This antibacterial activity coincided with significant reduction of lung weights (marker of organism-mediated pulmonary injury) in the CFDC group vs T/S and UC (p< 0.01). Survival was prolonged in the CFDC treatment group with 87% survival in comparison to that of T/S (25%) and UC (0%) (p< 0.01). Table 1. Efficacy of Cefiderocol in Experimental Stenotrophomonas maltophilia Pneumonia in Persistency Neutropenic Rabbits Conclusion Cefiderocol is highly active in treatment of experimental S. maltophilia pneumonia in persistently neutropenic rabbits, thus laying the foundation for future clinical investigations against this lethal infection. Disclosures Naoki Ishibashi, MD, Shionogi, Inc. (Employee) Benjamin Georgiades, n/a, Shionogi, Inc. (Consultant) Roger Echols, MD, Shionogi (Consultant) Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support) Yoshinori Yamano, PhD, Shionogi (Employee) Thomas J. Walsh, MD, PhD (hon), Scynexis (Consultant, Grant/Research Support)Shionogi (Consultant, Grant/Research Support)

Coexpression of Fungal Cell Wall-Modifying Enzymes Reveals Their Additive Impact on Arabidopsis Resistance to the Fungal Pathogen, Botrytis cinerea

The plant cell wall (CW) is an outer cell skeleton that plays an important role in plant growth and protection against both biotic and abiotic stresses. Signals and molecules produced during host–pathogen interactions have been proven to be involved in plant stress responses initiating signal pathways. Based on our previous research findings, the present study explored the possibility of additively or synergistically increasing plant stress resistance by stacking beneficial genes. In order to prove our hypothesis, we generated transgenic Arabidopsis plants constitutively overexpressing three different Aspergillus nidulans CW-modifying enzymes: a xylan acetylesterase, a rhamnogalacturonan acetylesterase and a feruloylesterase. The two acetylesterases were expressed either together or in combination with the feruloylesterase to study the effect of CW polysaccharide deacetylation and deferuloylation on Arabidopsis defense reactions against a fungal pathogen, Botrytis cinerea. The transgenic Arabidopsis plants expressing two acetylesterases together showed higher CW deacetylation and increased resistance to B. cinerea in comparison to wild-type (WT) Col-0 and plants expressing single acetylesterases. While the expression of feruloylesterase alone compromised plant resistance, coexpression of feruloylesterase together with either one of the two acetylesterases restored plant resistance to the pathogen. These CW modifications induced several defense-related genes in uninfected healthy plants, confirming their impact on plant resistance. These results demonstrated that coexpression of complementary CW-modifying enzymes in different combinations have an additive effect on plant stress response by constitutively priming the plant defense pathways. These findings might be useful for generating valuable crops with higher protections against biotic stresses.

Understanding Past and Present Vegetation Dynamics using the Palynological Approach: An Introductory Discourse

Palynology is a multi-disciplinary field of science that deals with the study and application of extinct, [fossilised] and extant palynomorphs (pollen and spore) and other related microscopic biological entities in the environment. It is divided into palaeo- and actuo-palynology, and provides substantial proxies to understanding past and present vegetation dynamics respectively. With reference to the two geological principles of uniformitarianism and of the evolution of fauna/flora, the distribution of plant indicators across ecological zones, palynomorph morphology and pollen analysis, palynology can be used to identify the change in past and present local and regional vegetation and climate and humans impact on the environment. Other supportive areas of endeavour like radiocarbon dating, sedimentology, taphonomic processes and geomorphology can be used to triangulate inferences drawn from palynological data. Palynomorphs are made of outer cell walls embedded with an inert, complex and resistant biopolymeric signature (called sporopollenin) which helps to facilitate long term preservation in different environmental matrices under favourable conditions, hence its widespread applicability. Palynology have proven to very reliable in reconstructing past vegetation, decrypting essential honeybee plants and understanding the impact of climate on plant population using pollen analysis, for which is the basis for the application of palynology in environmental studies. The application of palynology in climate, vegetation and anthropogenic studies begins with the selection of matrix (sediments from lake, river, ocean, excavation, relatively intact soil profile, bee products), coring or collection of samples, subjection to a series of chemically aided digestion, separation, physical filtration, decanting, accumulating of palynomorphs, microscopic study and ends with the interpretation of recovered information. Literature review on the application of palynology for understanding vegetation and climate interactions is presented in this paper.

DectiSomes: Glycan Targeting of Liposomal Drugs Improves the Treatment of Disseminated Candidiasis

Candida albicans causes life-threatening disseminated candidiasis. Individuals at greatest risk have weakened immune systems. An outer cell wall, exopolysaccharide matrix, and biofilm rich in oligoglucans and oligomannans help Candida spp. evade host defenses. Even after antifungal treatment, the one-year mortality rate exceeds 25%. Undoubtedly, there is room to improve drug performance. The mammalian C-type lectin pathogen receptors Dectin-1 and Dectin-2 bind to fungal oligoglucans and oligomannans, respectively. We previously coated amphotericin B-loaded liposomes, AmB-LLs, pegylated analogs of AmBisome, with the ligand binding domains of these two Dectins. DectiSomes, DEC1-AmB-LLs and DEC2-AmB-LLs, showed two distinct patterns of binding to the exopolysaccharide matrix surrounding C. albicans hyphae grown in vitro. Here we showed that DectiSomes were preferentially associated with fungal colonies in the kidneys. In a neutropenic mouse model of candidiasis, DEC1-AmB-LLs and DEC2-AmB-LLs delivering only one dose of 0.2 mg/kg AmB reduced the kidney fungal burden several fold relative to AmB-LLs. DEC1-AmB-LLs and DEC2-AmB-LLs increased the percent of surviving mice 2.5-fold and 8.3-fold, respectively, relative to AmB-LLs. Dectin-2 targeting of anidulafungin loaded liposomes, DEC2-AFG-LLs, and of commercial AmBisome, DEC2-AmBisome, reduced fungal burden in the kidneys several fold over their untargeted counterparts. The data herein suggest that targeting of a variety of antifungal drugs to fungal glycans may achieve lower safer effective doses and improve drug efficacy against a variety of invasive fungal infections.