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Watch out for bacterial leaf blight disease, farmers told
The Hindu Bureau TIRUCHI, DECEMBER 11, 2021 18:34 ISTUPDATED: DECEMBER 13, 2021 09:47 IST
Paddy crop with symptoms of bacterial leaf blight disease in a field near Pullampadi in Tiruchi district.
It has been found in standing paddy crop in some parts of Tiruchi district
With sporadic incidence of bacterial leaf blight disease in the standing samba and thaladi paddy crop in a few parts of the district, the Agriculture Department has advised farmers to take up appropriate measures to check its spread if symptoms appear in their fields.
Samba and thaladi paddy crop has been raised on about 49,000 hectares in the district. Due to the intermittent rains during the north-east monsoon and favourable climatic conditions, symptoms of bacterial leaf blight are appearing in the crop in some places. The bacterium enters through the cut wounds in the leaf tips and edges, becomes systemic and causes orange and yellow coloured wavy margins in the leaf tips and edges.
The disease symptoms first appear as small water-soaked translucent lesions on the edges of the leaf blade which later turn yellowish orange or brown, mostly confined to the edges of the leaf with wavy margins. As the disease progresses, the yellowish orange or brown lesions cover the entire leaf blade which may turn straw coloured .This will affect the photosynthesis of the plant, thereby reducing the yield.
For easy diagnosis, the leaf blade of the plant can be cut and dipped in water. If affected, white bacterial ooze could be noticed making the water turbid. Further, the bacterial infestation could lead to secondary infestation of the fungal pathogens which causes fungal diseases in the later stage of the crop.
Clipping of the tip of the seedling at the time of transplanting, heavy rain or dew, flooding, deep irrigation water, severe wind and application of excessive nitrogen, especially late top dressing are some of the favourable conditions for the spread of the disease.
If the disease is in the initial stage, 20% cow dung extract can be sprayed twice at 15 days interval. To control developed symptoms, 120 grams of streptomycin sulphate and tetracycline hydrochloride combination along with 500 grams of copper oxychloride mixed in 200 litres of water should be sprayed per acre. The spraying should be repeated 15 days later.
Farmers could contact the officials at the nearest Agricultural Extension Centres for more details and appropriate advice, Agriculture officials said.
HLB can infect an entire tree weeks before symptoms become apparent
Brazilian scientists have been able to measure the speed of a bacterium that causes the incurable citrus greening disease (Huanglongbing). HLB is the most devastating citrus disease in the world. Afflicted trees grow yellow leaves and low-quality fruit and eventually stop producing altogether.
Silvio A. Lopes, a plant pathologist based at Fundecitrus, research institution maintained by citrus growers of the State of Sao Paulo in Brazil: “We found that CLas can move at average speed of 2.9 to 3.8 cm per day. At these speeds a tree that is 3 meters in height will be fully colonized by CLas in around 80 to 100 days, and this is faster than the symptoms appear, which generally takes at least 4 months.”
Lopes and colleagues also studied the impact of temperature on the speed of colonization. They already knew that CLas does not multiply well in hot or cold environments, but now they have more specific data.
“We estimated that 25.7°C (78°F) was the best condition for CLas to move from one side to the other side of the tree,” said Lopes. This is the first time impact of temperature on plant colonization of CLas has been experimentally demonstrated. “The grower can use this information to select areas less risky for planting citrus trees.”
Novel Plantibodies show promise to protect citrus from Greening Disease
Citrus greening [huanglongbing (HLB)] has emerged as the most significant disease in citrus (Citrus sp.) agriculture. The disease is associated with the Candidatus Liberibacter species of bacteria. The most prevalent and virulent species in this group is Candidatus Liberibacter asiaticus. It is primarily vectored by the Asian citrus psyllid [ACP (Diaphorina citri)].
The bacteria and insect vector are present in many citrus orchards worldwide, including the United States, China, and Brazil. HLB often has a devastating impact on infected citrus; causing a rapid decline, with loss of fruit yield and quality and potentially leading to tree death. The bacteria has had a significant negative impact on the citrus industry, causing loss of fruit quality and yield, as well as loss of root mass, leading to tree decline. Finding a cure has been challenging due to the complexity of the CLas bacteria interactions with the citrus host and the Asian citrus psyllid. Another factor that has made it hard to recover from the disease is the tendency of the citrus industry to focus on a small number of cultivars with commercially desirable traits, but little genetic diversity.
Researchers who are working to find a citrus cultivar that is HLB resistant have a choice of either adding genetic variation through breeding with distant relatives or modifying the trees transgenically. In an article published this month in the Journal of the American Society for Horticultural Science, scientists present promising results from transgenic populations that produce antibodies that can bind with CLas proteins and reduce the bacteria’s ability to replicate.
This study advances the research needed to test the durability and strength of any resistance conferred by expression in rootstocks to a grafted tree and will hopefully lead to the development of a novel protection strategy for HLB.
According to Ed Stover, a Research Horticulturalist with the USDA Agricultural Research Service, “The Florida citrus industry desperately needs more HLB tolerant trees. If sufficient tolerance can be conferred by a single transgenic rootstock then it will greatly expedite implementation. Any transgenic solution will require extensive validation and analyses for non-target effects and food safety.”
by Weizmann Institute of Science A brown blotch on a plant leaf may be a sign that the plant’s defenses are hard at work: When a plant is infected by a virus, fungus or bacterium, its immune response keeps the disease from spreading by killing the infected cell, as well as a few surrounding ones. A new study at the Weizmann Institute of Science points to the evolutionary origins of this plant immune mechanism. The study may help explain how major plant defenses work and how they may one day be strengthened to increase resilience against plant diseases that each year cause billions of dollars of crop losses worldwide.
About two years ago, scientists in the United States and Australia discovered that when a plant’s immune system kills infected cells to contain disease, this action involves a protein with a segment called TIR that produces a certain signal molecule. In a new study led by Prof. Rotem Sorek of Weizmann’s Molecular Genetics Department and Dr. Gal Ofir, then a graduate student, Sorek’s team has revealed that bacteria also use TIR as an immune mechanism, and that TIR achieves immunity in plants and bacteria in similar ways.
SMART researchers develop method for early detection of bacterial infection in crops
Novel Raman Spectroscopy-based method enables early detection and quantification of pathogens in plants, which can impact plant disease management and agricultural industry Peer-Reviewed Publication
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IMAGE: RAPID DETECTION OF BACTERIAL INFECTION (XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS (XCC)) IN LEAFY VEGETABLE CHOY SUM USING QUANTITATIVE RAMAN SPECTROSCOPY-BASED ALGORITHM. ON THE RIGHT, THE INFECTION RESPONSE INDEX IS SHOWN, WHICH CAN AID FARMERS TO IDENTIFY INFECTIONS AND TAKE ACTION.view more CREDIT: SINGAPORE-MIT ALLIANCE FOR RESEARCH AND TECHNOLOGY (SMART)
Researchers from theDisruptive & Sustainable Technologies for Agricultural Precision (DiSTAP) Interdisciplinary Research Group (IRG) ofSingapore-MIT Alliance for Research and Technology (SMART), MIT’s research enterprise in Singapore and their local collaborators from Temasek Life Sciences Laboratory (TLL), have developed a rapid Raman spectroscopy-based method for the detection and quantification of early bacterial infection in crops. The Raman spectral biomarkers and diagnostic algorithm enable the non-invasive and early diagnosis of bacterial infections in crop plants, which can be critical for the progress of plant disease management and agricultural productivity.
Facing an increasing demand for global food supply and security, there is a growing need to improve agricultural production systems and increase crop productivity to overcome this challenge. Globally, bacterial pathogen infection in crop plants is one of the major contributors to agricultural yield losses. Climate change also adds to the problem by accelerating the spread of plant diseases. Hence, developing methods for rapid and early detection of pathogen-infected crops is important to improve plant disease management and reduce crop loss.
The breakthrough by SMART and TLL researchers offers a faster and more accurate method to detect bacterial infection in crop plants at an earlier stage, as compared to existing techniques. The team explained their research in a paper titled “Rapid detection and quantification of plant innate immunity response using Raman spectroscopy” published in the prestigious journal Frontiers in Plant Science.
“The early detection of pathogen-infected crop plants is a significant step to improve plant disease management,” says DiSTAP co-lead Principal Investigator Professor, TLL Deputy Chairman, and co-corresponding author, Chua Nam Hai. “It will allow the fast and selective removal of pathogen load and curb the further spread of disease to other neighbouring crops.”
Traditionally, plant diseases diagnosis involves a simple visual inspection of plants for disease symptoms and severity. “Visual inspection methods are often ineffective as disease symptoms usually manifest only at relatively later stages of infection when the pathogen load is already high, and reparative measures are limited. Hence, new methods are required for rapid and early detection of bacterial infection. The idea would be akin to having medical tests to identify human diseases at an early stage, instead of waiting for visual symptoms to show so that early intervention or treatment can be applied,” says DiSTAP Principal Investigator, MIT Professor, and co-corresponding author, Rajeev Ram.
While existing techniques, such as current molecular detection methods, can detect bacterial infection in plants, they are often limited in their use. Molecular detection methods largely depend on the availability of pathogen-specific gene sequences or antibodies to identify bacterial infection in crops; the implementation is also time-consuming and non-adaptable for on-site field application due to its high cost and bulky equipment required, making it impractical for use in agricultural farms.
“At DiSTAP, we have developed a quantitative Raman spectroscopy-based algorithm that can help farmers to identify bacterial infection rapidly. The developed diagnostic algorithm makes use of Raman spectral biomarkers and can be easily implemented in cloud-based computing and prediction platforms. It is more effective than existing techniques as it enables accurate identification and early detection of bacterial infection, both of which are crucial to saving crop plants that would otherwise be destroyed,” explained Dr Gajendra Pratap Singh, Scientific Director and Principal Investigator at DiSTAP, and co-lead author.
A portable Raman system can be used in agricultural farms and provides farmers with an accurate and simple yes or no response when used to test for the presence of bacterial infections in crop plants. The development of this rapid and non-invasive method will improve plant disease management and have a transformative impact on agricultural farms by efficiently reducing agricultural yield loss and increasing productivity.
“Using the diagnostic algorithm method, we experimented on several edible plants such as Choy Sum,” says DiSTAP and TLL Principal Investigator and co-corresponding author Dr Rajani Sarojam. “The results showed that the Raman spectroscopy-based method can swiftly detect and quantify innate immunity response in plants infected with bacterial pathogens. We believe that this technology will be beneficial for agricultural farms to increase their productivity by reducing their yield loss due to plant diseases.”
The researchers are currently working on the development of high-throughput, custom-made portable or hand-held Raman spectrometers that will allow Raman spectral analysis to be quickly and easily performed on field-grown crops.
The development and discovery of the diagnostic algorithm and Raman spectral biomarkers were done by SMART and TLL. TLL also confirmed and validated the detection method through mutant plants. The research is carried out by SMART and supported by the National Research Foundation of Singapore under its Campus for Research Excellence And Technological Enterprise (CREATE) programme.
The fast-spreading beech leaf disease is starting to kill the widespread, majestic American beech, which can rise to about 40 meters tall and live about 400 years.MIRCEA COSTINA/ALAMY STOCK PHOTO
A tree disease first spotted 9 years ago in Ohio is now a leading threat to one of eastern North America’s most important trees. The poorly understood malady, called beech leaf disease, is spreading rapidly and killing both mature American beeches and saplings, new research shows.
“This study documents how rapidly [the disease] has spread since its first observation in 2012,” says Robert Marra, a forest pathologist at the Connecticut Agricultural Experiment Station who was not involved with the work.
American beeches (Fagus grandifolia) are found across the eastern United States and Canada. The trees, which can grow nearly 40 meters tall and live up to 400 years, are a major player in many forests. Beeches constitute more than 25% of forests in Vermont, for example.
Historically, a blight called beech bark disease has been the primary threat to the species. But now, beech leaf disease appears to pose a bigger danger. First spotted in northeastern Ohio, it causes parts of leaves to turn leathery and branches to wither. The blight can kill a mature tree within 6 to 10 years. It has now been documented in eight U.S. states and in Canada.
In Rhode Island, observers first spotted beech leaf disease in 2020, confined to a small area, says Heather Faubert of the University of Rhode Island’s Plant Protection Clinic who was not involved with the study. But, “This year, it’s everywhere.”
Beech leaf disease causes some leaves to emerge with leathery, dark green parts. As the season continues, those parts may turn yellow or brown.MARY PITTS/ HOLDEN FORESTS AND GARDENS
To track the disease, Constance Hausman, an ecologist for a network of parks called the Cleveland Metroparks, and colleagues surveyed 64 0.04-hectare forest plots within 224 kilometers of Lake Erie in Ohio, Pennsylvania, New York, and Canada’s Ontario province. An analysis of 894 beeches in the plots found nearly half had the leaf disease, whereas just 34 had bark disease. Earlier surveys elsewhere had found the disease mostly attacked saplings, but the new work finds it is attacking mature trees, too, the team reported last month in Forest Ecology and Management. In forests near Lake Erie, beech leaf disease has now “become pervasive,” the group says.
The disease is “attacking the life cycle of beech trees in both directions,” Hausman says. The number of trees could fall so much in some forests that the species no longer serves key ecological functions, she warns, such as providing food and shelter for birds and other animals.
Another recent study by a different team examines an ongoing mystery: What exactly causes the disease? Earlier work raised suspicions that a tiny, previously unknown nematode worm that feeds on beech buds and leaves, dubbed Litylenchus crenatae mccannii, plays a role in spreading the blight.
Now, researchers report in Phytobiomes that when they examined diseased beech leaves, the tissues contained a fungus and four bacterial species also carried by the nematode. That suggests both the nematode and a pathogen it carries are contributing to the disease, says study co-author Pierluigi “Enrico” Bonello, an ecologist at Ohio State University, Columbus.
Marra is skeptical, however. He says one of the study’s suspects, Wolbachia, is known only to help its hosts. So he thinks its role in beech leaf disease, if any, might just be to strengthen the nematode’s attack.
So far, researchers haven’t identified a practical, cost-effective treatment for the disease, although some beeches appear to be resistant. But using those trees to breed new resistant strains could take decades, researchers say.
[Citrus greening (CG) is one of the most damaging diseases of the crops, affecting leaves and fruit. It is caused by fastidious phloem-inhabiting bacteria classified as _Candidatus_ Liberibacter asiaticus (CaLas; Asian greening; huanglongbing), africanus (including a subsp. capensis; African greening), or americanus (South American greening). The 3 pathogens can only be distinguished by molecular methods. Several phytoplasma species have been reported to cause symptoms similar to greening disease in citrus; coinfections of phytoplasmas with CaLas have also been recorded (see ProMED posts 20180214.5629251, 20190329.6392077). Further research is needed on symptomatology, epidemiology, and host impact of both single and mixed infections of these pathogens.
Symptoms include blotchy mottling and yellowing of leaves, as well as small, irregularly shaped fruits with a thick, pale peel and bad taste. Early symptoms may be confused with nutrient deficiencies. Affected trees become stunted, bear multiple off-season flowers, and may live for only a few years without ever bearing usable fruit. CG is restricted to _Citrus_ and close relatives because of the narrow host range of their psyllid vectors. The pathogens can also be spread by grafting and possibly by seed from infected plants or transovarially in the vectors. Both pathogens and vectors can be spread with plant material.
Disease management requires an integrated approach including use of clean planting and grafting stock, elimination of inoculum, use of pesticides for vector control in orchards, as well as chemical or biological control of vectors in non-crop reservoirs. Control using cultural methods, such as interplanting with non-host crops, is being trialled. In areas where a pathogen has not yet been detected, biological control of vectors has been used successfully to reduce insect numbers and, therefore, the risk of greening outbreaks (for example, see ProMED post 20090601.2034).
Antibiotics as leaf sprays, seed treatments, or trunk injections are being used occasionally to treat CG (see for example, ProMED posts 20181119.6154764, 20190320.6377319), but are subject to strict regulations in most countries due to their associated risks of facilitating the emergence of antibiotic resistances in other crop, animal, and human pathogens. Furthermore, beneficial soil microbes may be killed off as collateral damage, making the plants weaker and more susceptible to other diseases. Residues of antibiotics may also lead to rejection of exported produce by some countries.
In neighbouring India, CaLas was shown to be present in most states and widespread in all commercial citrus species and hybrids (ProMED post 20150409.3285806). While molecular diagnosis is often not obtained for local outbreaks, like the one reported above, CaLas seems to be the most likely CG pathogen involved in the region.
In South America, citrus in colder areas has been found less affected by CG (ProMED post 20201207.7999673), possibly due to vector insects in colder temperatures being less active or their numbers remaining lower. On the other hand, in Nepal, citrus psyllids have been found at increasing altitudes (ProMED post 20161129.4660906), potentially due to increasing overall temperatures there. This reflects similar effects observed for other pathogens and pests (for example, ProMED posts 20160902.4459660, 20160622.4302098, 20160509.4211696) migrating to new areas in many regions due to warming climates.
Farmers in Nubgang, Dekiling are cutting down orange trees for firewood and vegetable stalks. They are abandoning the cash crop and venturing into commercial farming or planting areca nut trees.
Oranges were the main source of income for the farmers in Nubgang. It was widely grown until citrus greening, one of the most serious citrus plant diseases in the world, emerged in the village, wiping out entire orchards. The dried orange trees remain abandoned in the orchards that were earlier filled with healthy fruiting trees.
Farmers say the problem became worse in the past three years.
Leki from Nubgang said that it was worrying to see all of the orange orchards fail in three years, as his family depended on the oranges for their livelihood.
“Living is becoming more difficult by the year. We lived a comfortable life because there were good returns from the oranges. Nothing is left now. There are no orange trees near home, or in the orchards,” he said.
He added that the family sold the orchard on contract to exporters. “We used to earn at least Nu 90,000 in a year. Exporters complained of a decreasing yield. We are not sure if they will pay us this time,” said Leki.
Most farms in Nubgang were filled with orange trees and the farmers owned separate orange orchards a few kilometres away from the village in the past. No orange trees can be seen today except for a few leafless and drying orange trees at a few homes.
Farmers said that there was no fruiting at all this time. A study was done and farmers were trained in managing orchards. However, the disease couldn’t be wiped out, as not all infected trees could be destroyed.
“The orange trees have started to die naturally. We have got no solution to this. It might also be because of soil fertility. The nature of the soil is different here. If we dig deep, we find sand and clay,” said Leki.
The farmer said that the officials from the agriculture sector have encouraged growing other cash crops. “It is equally discouraging when we don’t get the expected yield. It’s because of the poor soil quality. The orange trees might have died because of the soil,” said the farmer.
Another farmer from Nubgang said that they could replace the old trees with new seedlings, but it was not advisable because not all infected trees in the gewog have been destroyed.
“Now everyone has started growing areca nuts. Growing vegetables on a commercial scale is a challenge without a reliable water supply. We don’t have enough of a drinking water supply,” he said.
Former tshogpa from Nubgang, Dumber Singh Dahal, said that orange trees started to die in large numbers in 2014. “It will be difficult to earn income now. Some might choose to work as labourers for a living and move towards towns,” he said.
He added that the change in weather patterns could have also worsened the problem.
“There was no fruiting on time. Leaves started to drop and the whole tree dried up in two years. We tried to control the disease by spraying insecticides,” said Damber Singh.
Dekiling gewog agriculture extension officer, Sarita Rai said that the number of orange trees in the gewog fell every year. “It’s a nationwide problem caused mainly by poor orchard management. Farmers have to attend to other work and there are no good management practices,” she said.
She added that the agriculture sector, in collaboration with the agriculture research development centre (ARDC) in Samtenling, destroyed infected trees. It was not possible to cut down all of the trees because farmers were disappointed,” said Sarita Rai.
The farmers are encouraged to grow cash crops such as dragon fruit, ginger, and vegetables that are equally profitable. Orange production in Dekling has dropped every year, according to the official.
The gewog extension official said that it is challenging to procure insecticides, as it takes over six months to reach the farmers. The gewog also conducts awareness exercises on orchard management annually.
The official said that the gewog does not have the technical expertise to study the impact of the disease and works in collaboration with ARDC, Samtenling for technical supports and research.
Officials from the dzongkhag agriculture said that the citrus canopy management that focuses on improving orchard management practices and enhancing soil nutrients proved successful in Gakiling gewog.
While the disease couldn’t be wiped out without an advanced rehabilitation programme, the farmers were asked to replace orange trees with other fruit trees today.
Soil is a complex and competitive environment, forcing its inhabitants to develop strategies against competitors, predators, and pathogens. Identifying and understanding the molecular mechanisms has translational value for medicine, ecology, and agriculture. In this study, we show that a member of important soil-dwelling fungi (Mortierella) forms a tight alliance with toxin-producing bacteria (Mycoavidus) that live within the fungal hyphae and protect their host from nematode attack. This discovery is relevant since Mortierella species correlate with healthy soils and are used as plant growth–promoting fungi in agriculture. Unraveling an ecological role for fungal endosymbionts in Mortierella, our results contribute to the understanding of a mainspring in fungal–endobacterial symbioses and open the possibility for the development of new biocontrol agents.
Abstract
Fungi of the genus Mortierella occur ubiquitously in soils where they play pivotal roles in carbon cycling, xenobiont degradation, and promoting plant growth. These important fungi are, however, threatened by micropredators such as fungivorous nematodes, and yet little is known about their protective tactics. We report that Mortierella verticillata NRRL 6337 harbors a bacterial endosymbiont that efficiently shields its host from nematode attacks with anthelmintic metabolites. Microscopic investigation and 16S ribosomal DNA analysis revealed that a previously overlooked bacterial symbiont belonging to the genus Mycoavidus dwells in M. verticillata hyphae. Metabolic profiling of the wild-type fungus and a symbiont-free strain obtained by antibiotic treatment as well as genome analyses revealed that highly cytotoxic macrolactones (CJ-12,950 and CJ-13,357, syn. necroxime C and D), initially thought to be metabolites of the soil-inhabiting fungus, are actually biosynthesized by the endosymbiont. According to comparative genomics, the symbiont belongs to a new species (Candidatus Mycoavidus necroximicus) with 12% of its 2.2 Mb genome dedicated to natural product biosynthesis, including the modular polyketide-nonribosomal peptide synthetase for necroxime assembly. Using Caenorhabditis elegans and the fungivorous nematode Aphelenchus avenae as test strains, we show that necroximes exert highly potent anthelmintic activities. Effective host protection was demonstrated in cocultures of nematodes with symbiotic and chemically complemented aposymbiotic fungal strains. Image analysis and mathematical quantification of nematode movement enabled evaluation of the potency. Our work describes a relevant role for endofungal bacteria in protecting fungi against mycophagous nematodes.
A healthy soil nourishes plants and animals, purifies water and air, and promotes sustainable agriculture. Characteristic for highly complex and competitive soil ecosystems are the frequent and direct interactions between all soil-dwelling microorganisms, animals, and plants (1, 2), all of which need to be provided with minerals and carbon sources. Thus, carbon cycling, mainly promoted by fungal saprophytes and decomposers that release nutrients from decaying matter, plays a pivotal role for soil health (3, 4). Fungi belonging to the genus Mortierella are the most common soil-dwelling fungi, ubiquitously distributed in all parts of the world, inhabiting highly diverse niches including the rhizosphere and plant tissues (5⇓⇓⇓–9). Owing to their ability to degrade biopolymers as well as xenobiotics, they not only deliver energy-rich carbon sources but also clear the environment from pollutants (10, 11). Typically associated with healthy soils, Mortierella species are recognized as valuable plant growth–promoting fungi in agriculture (9, 12).
Even so, all fungi, including Mortierella species, are threatened by micropredators such as nematodes (13⇓–15). In order to oppose these predators, fungi have developed a diverse set of defense strategies. These include the production of toxic proteins and nematocidal natural products, hyphal piercing, trapping, egg parasitism, and endoparasitism (13, 16). Information on defense strategies employed by Mortierella species against nematodes is, however, scarce. It is known that Mortierella globalpina traps nematodes by means of its hyphae and penetrates the nematode’s cuticula. In this way, M. globalpina may protect its host plants from plant-parasitic nematodes (e.g., Meloidogyne chitwoodi) (17). Antinematode activities have been implicated for some Mortierella species (18, 19), including Mortierella alpina [against Meloidogyne javanica or Heterodera sp. (20, 21)], but it is not a general trait of Mortierella (21, 22). Apart from the hyphal trapping strategy, insight into the molecular basis of the antinematode activities of Mortierella is missing. Furthermore, on a more general note, it is remarkable that thus far no Mortierella secondary metabolites have been associated with potential protective roles against nematodes.
Here, we report a so far unknown strategy of a Mortierella species to protect itself from nematode attack. We provide evidence that cytotoxic benzolactones initially isolated from fungal cultures are in fact produced by bacterial endosymbionts that have been overlooked thus far. We also show that the bacteria dwelling in the fungal hyphae protect their host from predatory nematodes.
We reasoned that benzolactones CJ-12,950 and CJ-13,357 (Fig. 1A) (23) from cultures of Mortierella verticillata [synonym Podila verticillata (24)] could play a role as nematode defense metabolites. Although the initial report on CJ-12,950 and CJ-13,357 only stated that these compounds enhance the expression of the low-density lipoprotein receptor in human hepatocytes (23), they share the benzolactone enamide architecture with structurally related vATPase inhibitors (25, 26). Moreover, the architectures of CJ-12,950 and CJ-13,357 specifically resemble those of Burkholderia sp. strain B8 produced necroximes A to D (1 to 4), which proved to be cytotoxic (27). Since only the two-dimensional structures of CJ-12,950 and CJ-13,357 had been reported (23), we assigned their absolute configurations by examining the structural relationships with necroximes C and D. Optical rotation comparison, high-performance liquid chromatography (HPLC)–based coelution experiments and comparison of tandem mass spectrometry (MS/MS) fragmentation indicated that necroxime D (4) is identical to CJ-12,950, and necroxime C (3) is identical to CJ-13,357 (Fig. 1B and SI Appendix, Table S4). These assignments were corroborated by comparison of the NMR spectra of purified metabolites (SI Appendix, Table S9).
Bacterial origin of cytotoxic benzolactones from M. verticillata cultures. (A) Cytotoxic lactone compounds assigned to endofungal symbionts from the fungus R. microsporus (1–4), M. verticillata (3–4), Pseudomonas sp. (5), and a tunicate and the bacterium Gynuella sunshinyii (6). (B) Metabolic profiles of extracts from Burkholderia sp. strain B8 and M. verticillata NRRL 6337 as symbiont or cured strain as total ion chromatograms in the negative mode. (C) Fluorescence micrograph depicting endosymbionts living in the fungal hyphae; staining with Calcofluor White and Syto9 Green. (D) Phylogenetic relationships of Mortierella symbionts, Burkholderia sp. strain B8, and other bacteria based on 16S rDNA. BRE, Burkholderia-related endosymbiont of Mortierella spp. (E) Metabolic profiles of extracts from M. verticillata NRRL 6337 and other necroxime-negative M. verticillata strains analyzed for endosymbionts in this study as total ion chromatograms in the negative mode. M, medium component. (F) Growth of symbiotic M. verticillata NRRL 6337 in comparison to the cured strain.
Given the bacterial origin of the necroximes (27) and related benzolactones (25, 28⇓–30), we questioned the biosynthetic capability of M. verticillata and sought to identify the true producer. Since several Mortierella spp. have been reported to live in symbiosis with bacteria (31, 32), we suspected an endosymbiont to be the true source of 3 and 4. Yet, a 2018 report investigating the prevalence of Burkholderiaceae-related bacteria within Mortierella spp. stated that strain NRRL 6337 was devoid of endosymbionts (32). Nonetheless, we re-examined the same strain for endosymbionts by staining fungal hyphae with the chitin-binding Calcofluor White dye, and tentative endobacteria with the nucleic acid dye Syto9 Green (Fig. 1C). Fluorescence microscopy revealed the presence of endosymbiotic organisms in M. verticillata NRRL 6337 (SI Appendix, Fig. S1).
To identify the observed bacterial endosymbionts, we cut a small piece of fungal mycelium and extracted holobiont DNA, followed by PCR amplification of the 16S ribosomal DNA (rDNA) region using universal primers. Sequencing of the 16S rDNA region (SI Appendix, Table S1) and BLAST analysis indicated that the symbiont of M. verticillata NRRL 6337 is a Mycoavidus species. Notably, members of this genus have been reported as symbionts of soil-dwelling fungi (32). So far, the full genomes of only three Mycoavidus cysteinexigens strains from Mortierella elongata and Mortierella parvispora have been sequenced (31, 33⇓–35). PCR-amplified bacterial 16S rDNA sequences from other Mortierella fungi, however, revealed further Mycoavidus endosymbionts with three phylogenetically distant clades (Mortierella-associated Burkholderia-related endosymbiont [MorBRE] groups A to C) (32). Through phylogenetic analysis, we found that the Mycoavidus symbiont of M. verticillata NRRL 6337 falls into MorBRE group A (Fig. 1D and SI Appendix, Fig. S3) comprising symbionts of Mortierella humilis, Mortierella gamsii, Mortierella basiparvispora, and M. elongata (M. cysteinexigens). To better understand the occurrence of Mycoavidus endosymbionts in M. verticillata strains, we investigated five additional M. verticillata strains for the presence of endosymbionts. Amplification of the 16S rDNA regions from gDNA of symbionts of these strains revealed a conserved occurrence of Mycoavidus endosymbionts in M. verticillata strains. Interestingly, these additional endosymbionts all fall into another phylogenetic group together with Burkholderia sp. strain B8. Furthermore, analysis of the metabolic profiles of the respective fungi did not show any production of necroximes (Fig. 1 D and E). This finding shows that endosymbionts may frequently occur in Mortierella and other species of the order Mucorales, but they can be phylogenetically different.
To clarify whether bacterial endosymbionts are the true producers of 3 and 4, we aimed at curing M. verticillata NRRL 6337 of its symbiont through the addition of antibiotics (36). Over the course of several months, we subcultivated the fungal strain on agar plates containing kanamycin, ciprofloxacin, or chloramphenicol. During treatment, changes of the fungal growth were noticeable (Fig. 1F). Finally, we confirmed the absence of the symbionts by fluorescence staining, microscopic inspection, and PCR analysis (SI Appendix, Figs. S2 and S4). The metabolic profiling of the symbiont-free fungal strain by liquid chromatography (LC) combined with high-resolution electrospray ionization revealed the complete absence of 3 and 4 (Fig. 1B). These findings indicate that Candidatus Mycoavidus necroximicus is the true producer of the benzolactones.
Ca. M. necroximicus Dedicates 12% of Its Genome to Secondary Metabolism.
To gain insight into the symbiont’s biosynthetic potential, with particular focus on the molecular basis of necroxime biosynthesis, we aimed at sequencing the genome of the endosymbiont. Attempts to isolate and cultivate the endosymbiont in the absence of the fungal host, however, proved to be futile. Methods previously used to axenically cultivate similar fungal endobacteria did not enable growth of the endosymbionts (33, 37), indicating a strong dependence of the bacterial symbiont on the host environment. Thus, we sought to enrich the symbiotic bacteria for DNA isolation. Initially, physical disruption of the host’s mycelium resulted in high levels of contamination with fungal DNA, which complicated the assembly of the endosymbiont’s genome. Eventually, we succeeded in retrieving a bacterial cell pellet by filtration and centrifugation of the turbid supernatant of shaking cultures in baffled flasks and isolated the genomic DNA from resuspended bacteria.
The genome of the bacterial endosymbiont was sequenced using a combination of Oxford Nanopore MinION and Illumina NextSeq sequencing, and both data sets were used to generate a hybrid genome assembly. Of the 118 contigs, a single 2.4 Mb contig of putative bacterial origin was identified through homology searches using the Mycoavidus-like 16S rDNA sequence previously amplified from M. verticillata NRRL 6337. Following trimming of overlapping ends (suggesting a circular chromosome) the final 2.2 Mb contig was found to contain 1,768 CDS, 6 rRNAs, 42 tRNAs, and a GC content of 50.6% (genome accession number: PRJNA733818). The 16S rDNA sequence of the new strain has 98.82% nucleotide identity to M. cysteinexigens B1-EBT (33). Even so, genomic comparisons showed an average nucleotide identity of only 81.85% across the two genomes. By current standards for molecular species discrimination, the newly identified Mortierella endosymbiont should be considered a new species (Ca. M. necroximicus) (38, 39).
By comparative genomic analyses, we noted that the genomes of the two endofungal strains AG77 and B1-EBT isolated from M. elongata (33, 35) are 400 to 500 kb larger than the genome of Ca. M. necroximicus. Only the genome of strain B2-EB isolated from M. parvispora (34) is smaller (∼500 kb) than the genome of Ca. M. necroximicus (2.2 Mb). When investigating shared protein orthologs, we noted that a core genome encoding 1,164 proteins exists among the four genomes at the 70% identity level (Fig. 2A). However, a further all-versus-all comparison showed B1-EBT and AG77 to be the most closely related as they share ∼75% of their deduced proteome. The B2-EB and Ca. M. necroximicus strains are more distantly related to B1-EBT and AG77, as well as each other, with only a small number of proteins shared exclusively with either B1-EBT (20 and 17 proteins, respectively) or AG77 (17 and 22 proteins, respectively) (Fig. 2B).
Comparative genomic analyses of Mycoavidus spp. (A) Number of orthologous proteins among the four Mycoavidus strains at 70% identity. (B) Circos plot of shared protein orthologs, and secondary metabolite loci (detected by antiSMASH v5) in Mycoavidus genomes. Outer blocks (orange, brown, yellow, green) represent genome sizes, while the inner blocks represent genomic positions of secondary metabolite loci. Lines linking the three genomes show position of genes whose proteins are orthologous at 70% identity. Depicted are the genome sequences of M. cysteinexigens strains AG77, B1-EB, B2-EB, and Ca. M. necroximicus (Ca. M. nec.). (C) Number of gene clusters putatively coding for natural products in Mycoavidus spp. detected by antiSMASH and by manual assignment. (D) BGCs and their encoded assembly lines identified from the endofungal Ca. M. necroximicus are displayed. A, adenylation; AT, acyltransferase; C, condensation; DH, dehydratase; E, epimerization; Gnat, GCN5-related N-acetyltransferase; KR, ketoreductase; KS, ketosynthase; MT, methyltransferase; OX, oxygenase; TE, thioesterase domains. Acyl carrier (light blue) and peptidyl carrier proteins (dark blue) are shown as circles without designators. (E) Homologous benzolactone BGCs in the genome of Burkholderia strain B8 and Ca. M. necroximicus.
Whereas biosynthetic gene clusters (BGCs) are present in the genomes of all four studied Mortierella symbionts, antiSMASH analysis (40) revealed that the biosynthetic potential for secondary metabolites is by far the greatest in Ca. M. necroximicus (Fig. 2C). Despite the relatively small genome for Mycoavidus standards, ∼12% of its protein-encoding capacity is dedicated to natural product biosynthesis. We identified nine nonribosomal peptide synthetase (NRPS) gene clusters, two polyketide synthase (PKS) gene clusters, two hybrid PKS/NRPS gene clusters, and five other BGCs (Fig. 2D). Notably, several large PKS and NRPS gene loci present in the Ca. M. necroximicus genome are absent in the genomes of strains B1-EBT, B2-EB, and AG77 (Fig. 2B). This BGC list includes a cryptic BGC (Mcyst_0009–0017) encoding a PKS/NRPS hybrid that shows high similarity to the necroxime assembly line from Burkholderia sp. strain B8 (97% coverage, ∼70% amino acid identity), which has been unequivocally linked to necroxime biosynthesis by targeted gene knockouts (Fig. 2E) (27). The only major difference between the two BGCs is the NRPS gene necA, which is missing in the genome of Ca. M. necroximicus. This finding is in full agreement with the current biosynthetic model, since NecA is responsible for the attachment of the peptide side chain in 1 (Fig. 1A) (27), which is absent in 3 and 4. Furthermore, the architecture of the encoded PKS/NRPS modules is perfectly in line with the biosynthesis of the benzolactone enamide backbone of 3 and 4. Based on these in silico predictions, we inferred that this PKS/NRPS hybrid gene cluster codes for the biosynthesis of 3 and 4 (SI Appendix, Figs. S6 and S8). Together with the metabolic profiling of the cured fungal strain, these data indicate that the bacterial endosymbionts, not the fungus, are the true producers of the benzolactones 3 and 4. CJ-12,950 and CJ-13,357 are thus important additions to the small group of natural products that were believed to be fungal metabolites but are actually produced by bacterial endosymbionts; rhizoxins (41) and rhizonins (42) from symbionts of Rhizopus microsporus (43), and endolides from Stachylidium bicolor (44). From an ecological viewpoint, it is remarkabe that endosymbiotic bacteria were identified as the true producers of the virulence factor of the rice-seedling blight fungus R. microsporus (36, 37, 45). Given the different ecological context of Mortierella, however, we assumed that the necroximes may have another function in microbial interactions.
Necroximes Protect the Fungal Host from Nematode Attacks.
To learn more about the potential role of necroximes (3 and 4) in the ecological context of the Mortierella–Mycoavidus symbiosis, we investigated whether these toxins could impair the growth of, or even kill, competitors. Therefore, we considered that the common natural habitat of Mortierella species, including M. verticillata NRRL 6337, is soil, and that microbial survival in the soil environment is not only determined by the capacity to grow under harsh conditions but also by the ability to defend oneself from (micro)predators (46). Among the most abundant fungal predators are nematodes, which share the same soil habitat as Mortierella (47).
To determine if 3 and 4 or any other endobacteria-derived substance have anthelmintic activity, we first performed a viability assay against the model organism, Caenorhabditis elegans (48). We cultivated both cured (Mycoavidus-free) and symbiotic M. verticillata NRRL 6337 on potato dextrose agar (PDA agar). Cultures were extracted, and each extract was fractionated by preparative HPLC. The individual fractions (F1 to F9) were subsequently tested against C. elegans. Anthelmintic activity in this assay was determined by the ability of C. elegans to feed on a supplied Escherichia coli food source in the presence of the different fractions. Consumption of bacteria indicates unimpeded nematodes, whereas growth of E. coli indicates that the nematodes are negatively affected by the added substances (Fig. 3A). Notably, all fractions of the extract obtained from the cured strain culture were found to be inactive in the C. elegans assay. In contrast, we observed a marked nematocidal activity of fraction 6 from the extract of the symbiotic fungus. By LC/MS measurements we confirmed the presence of 3 and 4 in the active fraction. In order to determine the anthelmintic potency of the major metabolite 4, we performed the viability assay against C. elegans using increasing concentrations of the pure substance and determined an inhibitory concentration at 50% (IC50) value of 11.3 µg ⋅ mL−1 (24.66 µM) (Fig. 3B). Interestingly, the amount of isolated necroximes from fungal cultures grown on agar plates is ∼11 µg ⋅ mL−1. Assuming that the actual concentration in fungal hyphae is slightly higher due to an uneven diffusion into the agar and some loss during the purification steps, we conclude that the concentrations inside and around the fungal mycelium are sufficiently high to fully protect it from mycophagous nematodes.
Nematocidal activity of symbiont-derived toxins. (A) Viability assay of C. elegans in presence of extract fractions of symbiotic and cured M. verticillata NRRL 6337. HPLC profiles of extracts are shown with corresponding effect on nematodes, measured as effect on the E. coli optical density (OD). When nematode growth is impaired by the fraction, E. coli cells are not consumed, and thus the OD600 is not altered (error bars represent mean of three biological replicates). The red asterisk represents 4. (B) Toxicity screening of 4 against C. elegans. The red line marks IC50 at 11.3 µg ⋅ mL−1 (24.66 µM; 95% CI, 21.45 to 28.37 µM; error bars as mean of five biological replicates). (C) Nematode counts from propagation assay of M. verticillata and A. avenae cocultures. Bars represent relative nematode numbers compared to the mean of the nematode count from cured M. verticillata NRRL 6337 cultures. cur., cured; sym., symbiotic. *P < 0.02; ***P < 0.001; ****P < 0.0001. Data represent three biological replicates with three technical replicates each. (D) Workflow of image analysis and mathematical evaluation of A. avenae mobility in fungal–nematodal coincubations. Processing of time series is demonstrated by one time frame. Exemplary images of nematodes from two time frames (frame 1 and 26) are shown to illustrate differences in motility. Results of calculated mobility ratios (MR) were used for live or paralyzed/dead categorization. (E) Results of image analysis and mathematical quantification of nematode movement. Bars show ratio between moving/living nematodes and paralyzed/dead nematodes, which were harvested from cocultures of A. avenae with symbiotic M. verticillata NRRL 6337 cultures, cured NRRL 6337 cultures, or CSB 225.35 cultures. Numbers and error bars were calculated from minimal 176 worms from three biological replicates. (F) Stereomicroscopic images and schematic picture of chemical complementation assay with a magnitude of 25×. Sample of nematodes harvested from plates containing symbiotic, cured, or cured and with 4 chemically complemented M. verticillata NRRL 6337 cultures. (G) Schematic summary of tripartite interaction between fungal host, bacterial endosymbiont, and mycophagous nematodes.
In order to corroborate a potential host-protective role of the symbiont-derived toxin, we next focused on a fungivorous nematode. Therefore, we selected Aphelenchus avenae, a predator using a stylet to feed on fungi, which pierces the fungal cell wall and allows the fungivore to ingest the fungal cytoplasm (49, 50). Sharing the same soil habitat, A. avenae represents a realistic predator of Mortierella spp. (51). To investigate the effects of symbiotic and cured M. verticillata strains on the feeding behavior and survival of A. avenae, we determined the number of animals that were harvested from fungal–nematodal cocultures. In addition, we compared the mobility ratios of the nematodes in correlation to the presence or absence of the bacterial symbiont. As a control, we employed the symbiont-bearing, but necroxime-negative, M. verticillata strain CSB 225.35 (Fig. 1E), thus ruling out an influence solely based on the presence of bacterial symbionts.
To determine nematodal propagation rates, we inoculated plate cultures of symbiotic and cured fungi with A. avenae and cocultivated both organisms for 17 to 24 d (three biological triplicates). Subsequently, nematodes were isolated from the cocultivation plates by Baermann funneling (52), transferred onto water-agar plates, and counted by stereomicroscopic visualization. We found that significantly fewer nematodes are able to grow in the presence of the necroxime-producing endosymbionts (Fig. 3C and SI Appendix, Fig. S9 and Tables S5 and S6).
To scrutinize the effect of the toxin on the fitness of the fungivorous nematodes, we harvested the animals from cocultures and determined their movement—and thus the mobility ratios—by image analysis and mathematical quantification (Fig. 3D). Using stereoscopic time series to track their movement, we compared the area covered by each moving nematode during the time series to the area covered solely by its body without movement, allowing us to differentiate active (living) from inactive (dead or paralyzed) animals. Analyzing a minimum of 176 nematodes from three independent experiments, we observed a significant decrease in the mobility ratio of nematodes grown on symbiotic M. verticillata NRRL 6337 compared to cured NRRL 6337 cultures and to necroxime-negative CSB 225.35 cultures (Fig. 3E and SI Appendix, Fig. S10 and Tables S7 and S8).
HPLC analyses of the plate extracts detected necroximes only in symbiotic cultures of NRRL 6337 but not in cured strains or CBS 225.35, correlating once again the toxins with reduced numbers and lower fitness of the nematodes. To unambiguously assign the nematocidal activity in the propagation assay to the necroximes, we repeated the A. avenae assay with the cured Mortierella strain and chemically complemented the major toxin. Specifically, we overlaid the cured strain NRRL 6337 with solutions of 4 in increasing concentrations (25 µM [IC50], 50 µM, 109 µM, and 219 µM). We then compared A. avenae propagation in necroxime-complemented cultures to untreated cured as well as symbiotic fungi by microscopic examination after two weeks of coincubation. For cultures supplemented with 25 µM or 50 µM of 4, we noted a moderate reduction of nematode propagation, whereas in cultures supplemented with 109 µM or 219 µM of 4 the presence of nematodes in the fungus was abolished (Fig. 3F and SI Appendix, Figs. S11 and S12). The elevated concentrations compared to the IC50 value can be explained due to an uneven distribution of 4 into deeper layers of the hydrophobic fungal colony and the ongoing growth of fungal hyphae, which were not wetted with toxin solution. Nonetheless, these experiments unambiguously verified that the chemical complementation restores the anthelmintic effect. Thus, we uncovered an important role of a natural product in the complex tripartite interplay of symbiont, host, and (micro)predator (Fig. 3G).
Conclusions
In this study, we uncovered a previously overlooked bacterial endosymbiont that protects the important soil-dwelling fungus M. verticillata from a fungivorous nematode. Comparative genomics indicate that the yet unculturable bacterial symbionts belong to a new species that is endowed with a high biosynthetic potential. Through metabolic profiling of the symbiotic wild type and cured aposymbiotic fungi, we provide evidence that the endofungal bacteria are the true producers of highly toxic macrolides that were previously believed to be fungal metabolites. Importantly, these compounds (necroximes) efficiently protect the host from nematode attack, as demonstrated by coculture experiments, chemical complementation, and image analyses. Thus, this work not only reveals an ecological role of endofungal bacteria but also introduces a strategy to ward off micropredators. Consequently, the bacterial biosynthesis of necroximes provides an advantage of the fungal–bacterial alliance over other aposymbiotic or necroxime-negative symbiotic M. verticillata strains in the soil niche. Beyond inspiring the discovery of related tactics in symbioses, our findings may set the basis for new biocontrol agents, with the prospect of shielding plant hosts from plant-pathogenic nematodes.
Materials and Methods
Isolation of Natural Products.
For 3 and 4 isolation, M. verticillata NRRL 6337 was cultivated on PDA plates (Bacto, BD) at 26 °C. The culture was extracted twice with 1:1 volume of ethyl acetate overnight. The organic phase was concentrated under reduced pressure and the residue was dissolved in methanol. The extracts were prefractionated on an open Sephadex LH-20-column with methanol as eluent. Necroxime-containing fractions were further purified with a preparative HPLC under following conditions: A, H2O + 0.01% TFA; B, methanol; and 15 to 100% B in 35 min, 15 mL ⋅ min−1 [Phenomenex, Luna, 10 µm, C18(2), 100 Å, 250 × 21.2 mm]. NMR analysis was carried out on a 600 MHz Avance III Ultra Shield (Bruker), and signals were referenced to the residual solvent signal (DMSO-d6).
Identification of Endosymbionts in M. verticillata.
For the preparation of cured fungal strains, fungi were continuously subcultivated at 24 °C on PDA plates containing 40 µg ⋅ mL−1 ciprofloxacin or 50 µg ⋅ mL−1 kanamycin for several months. After phenotypic changes were observed by eye, an agar plate of each fungal culture was extracted with 20 mL ethyl acetate and controlled for the absence of 3 and 4 by LC/MS. Final verification of the cured fungal strains was performed by fluorescence staining (Calcofluor White Stain [Sigma] and SYTO 9 Green [Invitrogen]).
Genome Assembly for Ca. M. necroximicus.
M. verticillata NRRL 6337 was grown in MM9 medium (53) and orbitally shaken at 160 rpm and 26 °C. The turbid supernatant, containing bacteria from disrupted hyphae, was twice filtered through a membrane (pore diameter, 40 µm) and centrifuged (12,000 × g, 25 °C, 10 min) until a stabile pellet occurred. The genomic DNA was extracted with the MasterPure DNA Purification Kit (Epicentre). For long-read sequencing on the MinION platform, DNA quality was evaluated by pulsed-field gel electrophoresis and prepared for sequencing according to the protocol of the Ligation Sequencing kit (Oxford Nanopore). DNA was loaded onto a single MinION flow cell, and data were collected over a 72-h period. DNA was prepared for sequencing on the Illumina NextSeq platform using the Nextera XT DNA preparation kit (Illumina) with ×150 bp paired end chemistry and with a targeted sequencing depth of >50×. Combined MINion and Illumina sequencing data were assembled using the Unicycler hybrid assembler (54) to form a single contig 2.2 Mb containing a 98.82% match to the M. cysteinexigens rDNA gene. The evaluation of secondary metabolite loci was performed with antiSMASH version 5 (55).
Nematode Assays.
Liquid assays for active-fraction determination and potency assessment against C. elegans were conducted as previously described (48). For A. avenae coincubation assay, an aliquot of hyphae of each tested Mortierella strain was transferred to a PDA plate and incubated at 24 °C overnight. Nematodes were sterilized and starved. After one washing step with K-medium, nematodes were resuspended in 300 µL K-medium and aliquots of 50 µL were distributed onto the fresh fungal cultures. Plates were dried and controlled for living nematodes before they were incubated for 17 to 24 d at 20 °C. For the evaluation, nematodes were harvested via Baermann funneling (56). Funneled A. avenae were transferred on 1.5% water-agar plates containing 200 mM geneticin and 50 µg ⋅ mL−1 kanamycin overnight and subsequently monitored with a Zeiss Axio Zoom.V16 Stereomicroscope for worm count and bioinformatics (https://www.jipipe.org/). Remaining plates were extracted with ethyl acetate to control the metabolite production and processed as described before. For the A. avenae chemical complementation assay, an aliquot of hyphae of the respective fungus was transferred into 12-well plates filled with 1 mL PDA and incubated overnight. The 4 dissolved in 200 µL 50% MeOH was applied and evaporated at room temperature. Nematode suspensions of 50 µL were distributed onto the fungi, dried, and coincubated for 14 d at 20 °C. For evaluation, the coculture was removed from the well and washed in 5 mL K-medium overnight. The mixture was filtered through miracloth (Merck) to avoid agar carryover and left at 4 °C for 1 h. The remaining worms were transferred onto 6-well plates containing 5 mL 1.5% water-agar with 200 mM geneticin and 50 µg ⋅ mL−1 kanamycin. After the plates were dried, the worm count from each plate was assessed with a Zeiss Axio Zoom.V16 Stereomicroscope.
Data Availability
Genome sequence data have been deposited in GenBank (PRJNA733818). The 16S rDNA sequences of the Mortierella endosymbionts were deposited at the NCBI database (BRE_MvertCBS_346.66: MZ330684; BRE_MvertCBS_220.58: MZ330685; BRE_MvertCBS_225.35: MZ330686; BRE_MvertCBS_315.52: MZ330687; BRE_MvertCBS_100561: MZ330688).
Acknowledgments
Mortierella strains were supplied by ARS Culture Collection (NRRL) and the Jena Microbial Resource Collection. C. elegans was provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). A. avenae was received as a kind gift from Prof. Dr. M. Künzler (ETH Zürich). We thank E. Bratovanov (HKI) for helpful discussions. Assistance by K. Martin and S. Linde (HKI) is gratefully acknowledged. H.B. and S.P.N. were funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) Project No. 239748522 SFB 1127, the Cluster of Excellence “Balance of the Microverse,” and also the Leibniz Award (to C.H.). Z.C. and M.T.F. were funded by the DFG Project No. 316213987 SFB 1278 (Z01). I.R. acknowledges financial support from the European Union Horizon 2020 Research and Innovation Program under the Marie Sklodowska-Curie grant agreement No. 794343. R.G. was funded by the International Leibniz Research School for Microbial and Biomolecular Interactions Jena.
Footnotes
↵1H.B. and S.P.N. contributed equally to this work.
Author contributions: H.B., S.P.N., and C.H. designed research; H.B., S.P.N., K.V., Z.C., B.D., I.R., R.G., and S.J.P. performed research; H.B., S.P.N., K.V., Z.C., B.D., I.R., R.G., M.T.F., T.P.S., and S.J.P. analyzed data; and H.B., S.P.N., S.J.P., and C.H. wrote the paper.
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IMAGE: A DIVERSE CULTURE COLLECTION OF FUNGAL ISOLATES OBTAINED FROM AROUND THE WORLD HAS BEEN SCREENED BY RESEARCHERS AT LOS ALAMOS NATIONAL LABORATORY FOR POTENTIAL BACTERIAL ASSOCIATES.view more CREDIT: LOS ALAMOS NATIONAL LABORATORY
Los Alamos, N.M., Oct. 19, 2021 – In a novel, broad assessment of bacterial-fungal interactions, researchers using unique bioinformatics found that fungi host a remarkable diversity of bacteria, making bacterial-fungal interactions far more common and diverse than previously known.
“Until now, examples of bacterial-fungal interactions were pretty limited in number and diversity,” said Aaron Robinson, a biologist at Los Alamos National Laboratory and lead author of a new paper describing the research in Nature’s Communications Biology journal. “It had been assumed that bacterial-fungal associations might not be that common. But we found a lot of diverse bacteria that appear to associate with fungi, and we detected those associations at a frequent rate.”
The research contributes to an emerging understanding of the fungal bacteriome, the existence of bacteria both within and in close association with a fungal host, opening up possibilities for studying the interactions more intimately and connecting that research to issues such as ecosystem functioning and climate change impacts.
“This is a starting point to investigate mechanisms of bacterial-fungal interactions at a more intimate level,” said Robinson. “That research will be valuable for understanding what allows bacteria to associate with fungi, and how to best leverage that insight to accomplish goals for the Laboratory, for the Department of Energy, and for society in general. Understanding how these organisms interact with each other and contribute to larger systems is highly valuable in everything from modeling things like climate change to societally beneficial activities such as agricultural or industrial utilization of microbes.”
Researchers screened a total of 294 diverse fungal isolates from four culture collections from Europe, North America, and South America for potential bacterial associates. Collaborations with the Center for Integrated Nanotechnologies at Los Alamos allowed researchers to visually examine several of these associations using fluorescence in situ hybridization techniques.
These fluorescence microscopy examinations complemented the screening and confirmed the widespread and variable presence of bacterial associates among diverse fungal isolates and even within the hyphae (fungal tissue) of a single fungal host.
In addition to screening the culture collections, the research team also screened 408 fungal genome sequencing projects from the MycoCosm portal, a repository of fungal genome projects developed and maintained by the Department of Energy Joint Genome Institute.
Bacterial signatures were detected in 79 percent of the examined fungal genome projects. In multiple cases, the authors recovered complete or nearly complete genomes of these bacterial associates. Recovery of these fungal-associating bacterial genomes allowed for comparisons between fungal-associating and free-living bacteria.
Of the 702 total fungal isolates examined by the research team, bacterial associates were found in 88 percent—an unexpected detection rate relative to previous, more limited studies. The results shed light on the complexity and diversity of the fungal bacteriome across the fungal tree of life.
The study’s overview and description of diverse fungal-bacterial associations provides a path forward for understanding the associations in more depth. Continued analysis of the interactions will aid in a more complete understanding of environmental microbiome processes, particularly fungal and bacterial contributions to nutrient cycling, plant health and climate modeling.
Within the context of changing climate conditions, understanding how bacterial-fungal interactions impact plants, animals, and general ecosystem functioning in diverse environments and under diverse conditions, such as drought and warming, will also help predict and potentially manipulate the impacts of these interactions.
About Los Alamos National Laboratory Los Alamos National Laboratory, a multidisciplinary research institution engaged in strategic science on behalf of national security, is managed by Triad, a public service oriented, national security science organization equally owned by its three founding members: Battelle Memorial Institute (Battelle), the Texas A&M University System (TAMUS), and the Regents of the University of California (UC) for the Department of Energy’s National Nuclear Security Administration.
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Bacteria enters through natural openings at edges of corn leaves to cause Goss’s wilt
by American Phytopathological Society
Bacterial colonization and movement. Credit: Alexander Mullens and Tiffany M. Jamann
Goss’s bacterial wilt and leaf blight is one of the most damaging diseases affecting corn. The most effective way to control this disease is to plant corn varieties that are resistant to the disease. In other words, growers avoid the disease by growing certain varieties of corn. In part, this is the easiest method because scientists don’t yet know much about Goss’s wilt.
Alexander Mullens and Tiffany Jamann, two plant pathologists at the University of Illinois, set out to better understand the mechanics of this disease by following the causal pathogen. They genetically modified the pathogen so it would display green fluorescence, which made it easier to track the bacteria inside the plant. They were able to see how the bacteria entered the plant and where the bacteria congregated inside the leaf.
“While the bacteria had previously been known to enter the plants through wounds caused by wind or hail damage, we discovered that in the absence of damage it enters the leaf through natural openings at the edge of the leaf,” said Mullens. “Once in the plant, the bacteria are able to grow through the veins and exit the plant through natural pores in the leaf’s surface.” They also show that high concentrations of bacteria cause the freckles associated with Goss’s wilt.
They found that in resistant corn varieties, the bacteria aren’t able to grow as far from the entry site. “We can now use these tools to understand more about how different plant varieties restrict bacterial entry and growth,” said Jamann. “These tools will be useful in understanding how corn defends itself against this and other pathogens.”
Some of the most important pathogens in agriculture are vascular bacterial pathogens, like the causal pathogen of Goss’s wilt, so this is a good model to understand resistance to vascular plant diseases of all kinds.
More information: Alexander Mullens et al, Colonization and Movement of Green Fluorescent Protein-Labeled Clavibacter nebraskensis in Maize, Plant Disease (2020). DOI: 10.1094/PDIS-08-20-1823-RE Provided by American Phytopathological Society
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