Characterizing the Palm Pathogenic Thielaviopsis Species from Florida.

Thielaviopsis paradoxa sensu lato is a soilborne fungal pathogen that causes Thielaviopsis trunk rot and heart rot in palms. The loss of structural integrity resulting from trunk rot can cause the palm trunk to collapse suddenly and poses a serious threat to life and property. Even though rudimentary knowledge about the Thielaviopsis infection process in palms is available, nothing is known about the T. paradoxa species complex in the US. The aim of this study was to characterize T. paradoxa s. lat. isolates collected from diseased palms grown in Florida. Multi-locus phylogeny using three genes, ITS, ß-tubulin, and tef1-α, revealed that the isolates separate into two distinct clades with high bootstrap support. The majority of the isolates clustered with the species T. ethacetica, while two isolates formed a separate clade, distinct from T. musarum, and might represent an undescribed Thielaviopsis species. One representative isolate from each clade, when grown on three distinct media and at four different temperatures, showed differences in gross colony morphology, as well as growth rates. The T. ethacetica isolate TP5448 and the Thielaviopsis sp. isolate PLM300 grew better at opposite ends of the temperature spectrum tested in this study, i.e., 35 °C and 10 °C, respectively. In pathogenicity assays on whole plants, the T. ethacetica isolate proved to be more aggressive than Thielaviopsis sp. isolate PLM300, as it produced larger lesions when inoculated on wounded leaflets. An unequal distribution was observed for the mating-type locus of T. ethacetica, as 12 isolates carried the MAT1-1-1 allele, while the status for four isolates remained undefined. Variation in mycelial growth in response to different fungicides was also observed between the two clades. These results demonstrate the existence of two Thielaviopsis clades that can infect palms in Florida and underscore the need for targeted sampling to help uncover the diversity of Thielaviopsis species across palm-growing regions in the US.

Draft genome sequence of Streptomyces murinus strain SPC1 with antimicrobial potential against fungal pathogens of palms.

Streptomyces murinus strain SPC1, isolated from foxtail palm seeds, exhibited antimicrobial activity against fungal pathogens of palms. The assembled genome was 8.3 Mb, with 71.96% GC content, and contained 37 secondary metabolite clusters (SMCs). A complete SMC for antifungal metabolite pentamycin (fungichromin) biosynthesis was identified in SPC1 genome.

Nitrogen and phosphorous acquisition strategies drive coexistence patterns among archaeal lineages in soil.

Soil represents the largest reservoir of Archaea on Earth. Present-day archaeal diversity in soils globally is dominated by members of the class Nitrososphaeria. The evolutionary radiation of this class is thought to reflect adaptations to a wide range of temperatures, pH, and other environmental conditions. However, the mechanisms that govern competition and coexistence among Nitrososphaeria lineages in soil remain poorly understood. Here we show that predominant soil Nitrososphaeria lineages compose a patchwork of gene inventory and expression profiles for ammonia, urea, and phosphate utilization. In contrast, carbon fixation, respiration, and ATP synthesis genes are conserved and expressed consistently among predominant phylotypes across 12 major evolutionary lineages commonly found in soil. In situ gene expression profiles closely resemble pure culture reference strains under optimal growth conditions. Together, these results reveal resource-based coexistence patterns among Nitrososphaeria lineages and suggest complementary ecophysiological niches associated with differential nutrient acquisition strategies among globally predominant archaeal lineages in soil.

Ganoderma zonatum causes butt rot of areca (Dypsis lutescens) and robellini (Phoenix roebelenii) palms in Florida.

Ganoderma butt rot is a lethal disease of palms (Arecaceae) prevalent in palm-growing regions in the US that infects at least 58 species of palms (Elliott and Broschat 2001). Early symptoms appear as wilting of older fronds in the lower part of the canopy, and as disease progresses, wilting advances to younger leaves higher in the canopy towards the unopened spear leaf eventually killing the palm. A characteristic sign of the disease is the appearance of fruiting bodies (basidiomata) at the base of the palm trunk close to soil line. Ganoderma butt rot disease was detected on clustering palm species, areca palms, with 9 (82%) clusters showing Ganoderma basidiocarps and dead stumps, and mortality was observed in 5 (45%) clusters. A sterile scalpel was used to transfer the context tissue from Ganoderma basidiomata to full-strength potato dextrose agar selective media supplemented with streptomycin (100 mg/l), lactic acid (2 ml/l) and benomyl (4 mg/l). The pure culture for isolate GAN-33 was grown at 28°C in complete darkness for 10 days. The fungal colony was ivory white in color that grew radially as a dense mycelial mat without any sporulation. To establish the identity of the fungus, DNA was extracted using the Qiagen DNeasy PowerSoil kit (Cat. #12888). Three barcoding genes, nuclear ribosomal DNA internal transcribed spacer (ITS) region, RNA polymerase II subunit 2 (rpb2) and translation elongation factor 1α (tef1α) were amplified using primers ITS1/ITS4 (White et al 1990), bRPB2-6f/bRPB2-b7.1R (Matheny et al 2007) and EF1-983F/EF1-2212R (Matheny et al 2007), respectively. The sequences were deposited in GenBank, accession numbers KX853442, KX853466 and KX853491 for ITS, rpb2 and tef1α, respectively (Elliott et al 2018). Comparison to the NCBI nucleotide sequence database identified isolate GAN-33 as Ganoderma zonatum based on 100, 99 and 99% similarity to ITS, rpb2 and tef1α sequences, respectively. Pathogenicity of G. zonatum isolate GAN-33 was determined on 1-year old seedlings of areca palm (Dypsis lutescens) and pygmy date palm (Phoenix roebelenii). Ganoderma zonatum inoculum was prepared by transferring two-week old cultures to autoclaved wheat berries and allowed to colonize for two weeks. Seedlings were gently removed from the pot and the roots were trimmed before placing them back in the pot ensuring that the roots were in contact with the G. zonatum colonized wheat berries. The inoculated and control seedlings were maintained in a growth chamber at 28°C 60% RH (daytime) and 24°C 50% RH (night time), 12h:8h light:dark period, and watered twice a week. Initial wilting symptoms started appearing approximately one month after inoculation and mortality was observed for a total of four seedlings at three months after inoculation i.e., 2 out of 3 G. zonatum inoculated seedlings died for both areca and robellini palms, whereas the non-inoculated areca and robellini palm control seedlings remained healthy and alive. The pathogen was re-isolated from inoculated roots, and its identity was confirmed by colony morphology and PCR using G. zonatum specific primers (Chakrabarti et al 2022). To the best of our knowledge this is the first report establishing G. zonatum as the pathogen responsible for Ganoderma butt rot of palms.

Year-around survey and manipulation experiments reveal differential sensitivities of soil prokaryotic and fungal communities to saltwater intrusion in Florida Everglades wetlands.

Global sea-level rise is transforming coastal ecosystems, especially freshwater wetlands, in part due to increased episodic or chronic saltwater exposure, leading to shifts in biogeochemistry, plant- and microbial communities, as well as ecological services. Yet, it is still difficult to predict how soil microbial communities respond to the saltwater exposure because of poorly understood microbial sensitivity within complex wetland soil microbial communities, as well as the high spatial and temporal heterogeneity of wetland soils and saltwater exposure. To address this, we first conducted a two-year survey of microbial community structure and bottom water chemistry in submerged surface soils from 14 wetland sites across the Florida Everglades. We identified ecosystem-specific microbial biomarker taxa primarily associated with variation in salinity. Bacterial, archaeal and fungal community composition differed between freshwater, mangrove, and marine seagrass meadow sites, irrespective of soil type or season. Especially, methanogens, putative denitrifying methanotrophs and sulfate reducers shifted in relative abundance and/or composition between wetland types. Methanogens and putative denitrifying methanotrophs declined in relative abundance from freshwater to marine wetlands, whereas sulfate reducers showed the opposite trend. A four-year experimental simulation of saltwater intrusion in a pristine freshwater site and a previously saltwater-impacted site corroborated the highest sensitivity and relative increase of sulfate reducers, as well as taxon-specific sensitivity of methanogens, in response to continuously pulsing of saltwater treatment. Collectively, these results suggest that besides increased salinity, saltwater-mediated increased sulfate availability leads to displacement of methanogens by sulfate reducers even at low or temporal salt exposure. These changes of microbial composition could affect organic matter degradation pathways in coastal freshwater wetlands exposed to sea-level rise, with potential consequences, such as loss of stored soil organic carbon.

Multilocus, Multiplex Detection of Ganoderma zonatum from Environmental Samples.

Ganoderma butt rot of palms is caused by a white rot basidiomycete fungus, Ganoderma zonatum. Typical symptoms include wilting of fronds that starts in the lower canopy and moves to the top. As wilting symptoms are also associated with other diseases and disorders, appearance of basidiomata on the trunks is necessary to confirm this disease. Basidiomata develop late in the disease cycle, making early diagnostics challenging. Here, we describe a DNA-based molecular diagnostic assay that could be used to confirm the presence of G. zonatum in palm trunks before conks are observed. Primers tailored to end on single-nucleotide polymorphisms (SNPs), that differentiate G. zonatum from 14 other Ganoderma taxa, were designed from multiple regions in four genes: internal transcribed spacer (ITS), RNA polymerase 1 (rpb1), rpb2, and translation elongation factor 1-α (tef1-α). A set of three primer pairs could successfully determine the incidence of G. zonatum with high specificity and sensitivity in different environmental samples such as sawdust collected from naturally infected palm trunks and soil samples containing G. zonatum basidiospores. This rapid PCR-based assay could potentially be used to detect inoculum sources of the fungus and track its movement and survival in different palm tissues and environments. Early detection of G. zonatum is a crucial step toward building and implementing better disease management strategies and mitigating potential risks from palm failures due to decay.

Cytotoxicity of Medicinal Mushrooms Oudemansiella canarii and Ganoderma lucidum (Agaricomycetes) Against Hematologic Malignant Cells via Activation of Apoptosis-Related Markers.

Cancer is the second leading cause of death worldwide, and despite of the of the availability of the advanced chemical treatments, development of effective and safe alternatives derived from natural resources are still of high interest. Mushroom is one of the important resources of pharmacologically active cytotoxic compounds. In this paper, we report the cytotoxicity of ethanolic extracts of Oudemansiella canarii (Jungh.) Höhn. and Ganoderma lucidum (W. Curt.: Fr.) P. Karst. against nine hematologic malignant cells and describe their molecular mechanisms. Cell lines were exposed to varying concentrations of mushroom extracts for 48 h and the cell proliferation and apoptosis parameters were determined. Western blot analysis was performed to determine the extract-induced changes in the level of apoptosis-related proteins in cancer cell lines and patient-derived mononuclear cells. Results revealed that O. canarii and G. lucidum extracts exhibited cytotoxicity with IC50 values of 26.8-66.0 ppm and 48.1-78.4 ppm, respectively, in all the cancer cell lines used. Mushroom extracts inhibited cell proliferation by 57.3-72.5% (O. canarii) and 44.2-67.4% (G. lucidum), which correlates to the activation of apoptosis as indicated by increased annexin V positivity, cells in sub G0/G1 phase and production of reactive oxygen species, and decreased mitochondrial membrane potential. Western blot analysis showed increase in the level of apoptotic markers (cleaved PARP1, cleaved caspase 3 and phosphorylation of histone 2AX) and activation of the stress-activated protein kinase (SAPK/JNK) signaling pathway. The extract-activated apoptosis was also observed in mononuclear cells isolated from the peripheral blood of leukemia and lymphoma patients. In conclusion, activation of pro-apoptotic markers is one of the major mechanisms of the cytotoxicity of O. canarii and G. lucidum extracts against hematologic malignant cells.

Nutrient-Limited Enrichments of Nitrifiers From Soil Yield Consortia of Nitrosocosmicus-Affiliated AOA and Nitrospira-Affiliated NOB.

The discovery of ammonia-oxidizing archaea (AOA) and complete ammonia-oxidizing (comammox) bacteria widespread in terrestrial ecosystems indicates an important role of these organisms in terrestrial nitrification. Recent evidence indicated a higher ammonia affinity of comammox bacteria than of terrestrial AOA and ammonia-oxidizing bacteria (AOB), suggesting that comammox bacteria could potentially represent the most low-nutrient adapted nitrifiers in terrestrial systems. We hypothesized that a nutrient-limited enrichment strategy could exploit the differences in cellular kinetic properties and yield enrichments dominated by high affinity and high yield comammox bacteria. Using soil with a mixed community of AOA, AOB, and comammox Nitrospira, we compared performance of nutrient-limited chemostat enrichment with or without batch culture pre-enrichment in two different growth media without inhibitors or antibiotics. Monitoring of microbial community composition via 16S rRNA and amoA gene sequencing showed that batch enrichments were dominated by AOB, accompanied by low numbers of AOA and comammox Nitrospira. In contrast, nutrient-limited enrichment directly from soil, and nutrient-limited sub-cultivation of batch enrichments consistently yielded high enrichments of Nitrosocosmicus-affiliated AOA associated with multiple canonical nitrite-oxidizing Nitrospira strains, whereas AOB numbers dropped below 0.1% and comammox Nitrospira were lost completely. Our results reveal competitiveness of Nitrosocosmicus sp. under nutrient limitation, and a likely more complex or demanding ecological niche of soil comammox Nitrospira than simulated in our nutrient-limited chemostat experiments.

Cytotoxicity of Gymnopilus purpureosquamulosus extracts on hematologic malignant cells through activation of the SAPK/JNK signaling pathway.

Treatment of hematologic malignancies is a formidable challenge for hematologists and there is an urgent need to identify safe and efficacious agents either via synthesis in the laboratory or isolation from natural products. Here, we report the cytotoxicity of extracts from mushroom Gymnopilus purpureosquamulosus Høil (G. pps) and describe its molecular mechanisms. Using leukemia, lymphoma and multiple myeloma cell lines, 28-35 ppm G. pps extract inhibited cell proliferation by ~46-79%, which correlates with activation of apoptosis as indicated by increase in annexin V-positive cells (~5-8-fold), production of reactive oxygen species (~2-3-fold), cells in sub G0/G1 phase (~3-13-fold), caspase 3 enzymatic activity (~1.6-2.9-fold), DNA fragmentation, PARP1 cleavage and down-regulation of prosurvival proteins. Mitochondrial membrane potential decreased and leakage of pro-apoptotic factors to cytoplasm was observed, consistent with the activation of intrinsic apoptosis. Western blot analysis showed activation of the ASK1-MEK-SAPK/JNK and ASK1-P38 MAPK pathways possibly due to changes in the cellular redox status as suggested by decreased protein levels of peroxiredoxin, thioredoxin and thioredoxin reductase. Moreover, antioxidant N-acetylcysteine alleviated the cytotoxicity of G. pps. Pharmacological inhibition of SAPK/JNK and P38 alleviated the G. pps-mediated cytotoxicity. The extract activated apoptosis in leukemia and lymphoma patient cell samples but not in mononuclear cells from healthy donors further supporting the therapeutic values of G. pps for hematologic malignancies.

Ammonia-oxidizing archaea are integral to nitrogen cycling in a highly fertile agricultural soil.

Nitrification is a central process in the global nitrogen cycle, carried out by a complex network of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), complete ammonia-oxidizing (comammox) bacteria, and nitrite-oxidizing bacteria (NOB). Nitrification is responsible for significant nitrogen leaching and N2O emissions and thought to impede plant nitrogen use efficiency in agricultural systems. However, the actual contribution of each nitrifier group to net rates and N2O emissions remain poorly understood. We hypothesized that highly fertile agricultural soils with high organic matter mineralization rates could allow a detailed characterization of N cycling in these soils. Using a combination of molecular and activity measurements, we show that in a mixed AOA, AOB, and comammox community, AOA outnumbered low diversity assemblages of AOB and comammox 50- to 430-fold, and strongly dominated net nitrification activities with low N2O yields between 0.18 and 0.41 ng N2O-N per µg NOx-N in cropped, fallow, as well as native soil. Nitrification rates were not significantly different in plant-covered and fallow plots. Mass balance calculations indicated that plants relied heavily on nitrate, and not ammonium as primary nitrogen source in these soils. Together, these results imply AOA as integral part of the nitrogen cycle in a highly fertile agricultural soil.

The Seagrass Holobiont and Its Microbiome.

Seagrass meadows are ecologically and economically important components of many coastal areas worldwide. Ecosystem services provided by seagrasses include reducing the number of microbial pathogens in the water, providing food, shelter and nurseries for many species, and decreasing the impact of waves on the shorelines. A global assessment reported that 29% of the known areal extent of seagrasses has disappeared since seagrass areas were initially recorded in 1879. Several factors such as direct and indirect human activity contribute to the demise of seagrasses. One of the main reasons for seagrass die-offs all over the world is increased sulfide concentrations in the sediment that result from the activity of sulfate-reducing prokaryotes, which perform the last step of the anaerobic food chain in marine sediments and reduce sulfate to H2S. Recent seagrass die-offs, e.g., in the Florida and Biscayne Bays, were caused by an increase in pore-water sulfide concentrations in the sediment, which were the combined result of unfavorable environmental conditions and the activities of various groups of heterotrophic bacteria in the sulfate-rich water-column and sediment that are stimulated through increased nutrient concentrations. Under normal circumstances, seagrasses are able to withstand low levels of sulfide, probably partly due to microbial symbionts, which detoxify sulfide by oxidizing it to sulfur or sulfate. Novel studies are beginning to give greater insights into the interactions of microbes and seagrasses, not only in the sulfur cycle. Here, we review the literature on the basic ecology and biology of seagrasses and focus on studies describing their microbiome.

Persistence of exotic Newcastle disease virus (ENDV) in laboratory infected Musca domestica and Fannia canicularis.

House flies (Musca domestica) and little house flies (Fannia canicularis) were examined for their ability to take up and harbor a velogenic strain of exotic Newcastle disease virus (ENDV) (family Paramyxoviridae, genus Avulavirus). Laboratory-reared flies were allowed to feed on evaporated milk containing ENDV at a virus concentration of 10(8.3) egg infectious dose (EID)50/0.1 ml or on poultry feces containing an ENDV titer of 10(5.8) EID50/0.1 g. Flies exposed to either infectious food source for 24 hr became transiently infected with virus. Virus persisted predominantly in the mid- and hindgut, with relatively little virus isolated from the remainder of the fly body. Virus persisted similarly in both fly species that were fed evaporated milk containing ENDV, with a maximum ENDV titer of 10(5.98) EID50/fly for the house fly and 10(4.78) EID50/fly for the little house fly at 1 day postexposure; titers decreased on subsequent days to 10(2.38) EID50/fly for house fly and > or = 1 EID50/fly for little house fly at 5 days postexposure. Both fly species acquired viral titers greater than the infective dose for a susceptible chicken (10(3.0) EID50-10(4.0) EID50). In addition, flies fed evaporated milk containing a high titer of ENDV maintained viral titers above the infective dose for up to 4 days postexposure to the infectious food source. Flies fed on infective feces retained a chicken infective dose for only one day. The decrease in viral titer over time was significantly explained by logistic regression for both fly species (P < 0.05). The slope of the regression line was not different for the two fly species (P < 0.05), indicating a similar rate of virus loss.

Detection and isolation of exotic Newcastle disease virus from field-collected flies.

Flies were collected by sweep net from the vicinity of two small groups of "backyard" poultry (10-20 chickens per group) that had been identified as infected with exotic Newcastle disease virus (family Paramyxoviridae, genus avulavirus, ENDV) in Los Angeles County, CA, during the 2002-2003 END outbreak. Collected flies were subdivided into pools and homogenized in brain-heart infusion broth with antibiotics. The separated supernatant was tested for the presence of ENDV by inoculation into embryonated chicken eggs. Exotic Newcastle disease virus was isolated from pools of Phaenicia cuprina (Wiedemann), Fannia canicularis (L.), and Musca domestica L., and it was identified by hemagglutination inhibition with Newcastle disease virus antiserum. Viral concentration in positive pools was low (<1 egg infectious dose50 per fly). Isolated virus demonstrated identical monoclonal antibody binding profiles as well as 99% sequence homology in the 635-bp fusion gene sequence compared with ENDV recovered from infected commercial egg layer poultry during the 2002 outbreak.