Research Articles (Forestry and Agricultural Biotechnology Institute (FABI))

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Molecular mechanisms underlying tree host-pathogen interactions under drought stress and subsequent rewatering in Eucalyptus grandis
(Elsevier, 2024-12-02) Teshome, Demissew Tesfaye; Zharare, Godfrey Elijah; Ployet, Raphael; Naidoo, Sanushka; sanushka.naidoo@up.ac.za
Abiotic stresses such as drought change plant-pathogen interactions by affecting both hosts and pathogens. Here, we aimed to unravel the molecular mechanisms underlying forest tree-pathogen interactions under drought stress and subsequent rewatering. We conducted glasshouse experiments involving infection by the stem canker-causing fungal pathogen Chrysoporthe austroafricana under drought stress and rewatering in Eucalyptus grandis and investigated host and pathogen transcriptomic changes using RNA-seq data from our current combined stress experiment as well as previous single stress studies. We found that mild drought stress enhances disease progression while, upon rewatering, pathogen infection delays recovery of leaf stomatal conductance. Transcriptomic changes in the host support increased susceptibility to the pathogen while the in planta fungal transcriptome suggests prioritization of survival in the drought-stressed host. Upon rewatering, changes in the host transcriptome suggest allocation of resources to stress responses at the expense of growth and carbohydrate storage while that of the pathogen indicate downregulation of some fungal metabolic pathways potentially because the pathogen takes advantage of changes in the host. Our study identified key molecular processes and genes that provide mechanistic insights into tree-pathogen interactions under abiotic stresses. This enables prediction of tree resilience under a changing climate and contributes towards future tree health improvement endeavours.
Mulching of post-harvest residues and delayed planting improves fungal biodiversity in South African Eucalyptus plantations and enhances plantation productivity
(Elsevier, 2025-06) Bose, Tanay; Roux, Jolanda; Titshall, Louis; Dovey, Steven B.; Hammerbacher, Almuth; tanay.bose@fabi.up.ac.za
Short-rotation Eucalyptus plantations provide essential forest products, with productivity and soil health influenced by residue management and planting strategies. This study examined the effects of burning or mulching post-harvest residue followed by immediate versus delayed planting on soil fungal biodiversity, soil properties, and tree growth across four sites in KwaZulu-Natal, South Africa. Plots were planted either three months ('immediate') or six months ('delayed') after treatment implementation. Volume measurements assessed tree growth, and soil attributes, including moisture, temperature, and nutrient levels, were analyzed. Soil samples were collected in November 2019 and March 2022, and fungal communities were analyzed through high-throughput sequencing targeting the internal transcribed spacer 1 (ITS1) region. Data emerging from this study showed mulched plots had significantly higher tree volume, with delayed planting increasing productivity by 13.6 % at 24–36 months and 25 % at 36–48 months post-planting. Soil moisture was 1.3–2 times higher in mulched plots than in burnt plots. Mulching significantly reduced the maximum soil temperatures by 4.5–6.8 °C. Four months after treatment, burnt plots had higher pH (1.1-fold), carbon (2.2-fold), phosphate (1.6-fold) and manganese (2.5-fold). Initially, mulched plots had lower fungal biodiversity (0.81-fold) than burnt plots but surpassed them after 28 months (1.28-fold increase). Fungal community overlap declined from 83.28 % to 40.64 %, with mulching supporting higher saprotroph (1.3-fold) and symbiotroph (1.25-fold) abundances, while delayed planting increased pathotroph presence by 1.5-fold in burnt plots. These findings highlight the long-term benefits of mulching and delayed planting in enhancing fungal biodiversity, promoting beneficial microbial communities, and improving tree growth, contributing to more sustainable Eucalyptus plantation management.
Mesorhizobium salmacidum sp. nov. and Mesorhizobium argentiipisi sp. nov. are symbionts of the dry-land forage legumes Lessertia diffusa and Calobota sericea
(Springer, 2025-03) Muema, Esther K.; Van Lill, Melandre; Venter, S.N. (Stephanus Nicolaas); Chan, Wai Yin; Claassens, Ricu; Steenkamp, Emma Theodora
Legumes Lessertia diffusa and Calobota sericea, indigenous to South Africa, are commonly used as fodder crops with potential for sustainable livestock pasture production. Rhizobia were isolated from their root nodules grown in their respective soils from the Succulent Karoo biome (SKB) in South Africa, identified and characterized using a polyphasic approach. Sequence analysis of the 16S rRNA gene confirmed all isolates as Mesorhizobium members, which were categorized into two distinct lineages using five housekeeping protein-coding genes. Lineage I included 14 strains from both legumes, while Lineage II comprised a single isolate from C. sericea. Differences in phenotypic traits were observed between the lineages and corroborated by average nucleotide identity analyses. While all strains nodulated their original hosts, strains from C. sericea failed to effectively nodulate L. diffusa and vice versa. Phylogenetic analyses of nitrogen fixation (nifH) and nodulation (nodA, nodC) loci grouped all strains in a single clade, suggesting that unique symbiotic loci determine nodulation of these legumes. We designated Lineage I and II as Mesorhizobium salmacidum sp. nov. (Ld1326Ts; GCA_037179605.1Ts) and Mesorhizobium argentiipisi sp. nov. (Cs1330R2N1Ts; GCA_037179585.1Ts), using genome sequences as nomenclatural types according to the Nomenclatural Code for Prokaryotes using Sequence Data, thus avoiding complications with South Africa's biodiversity regulations. Identifying effective microsymbionts of L. diffusa and C. sericea is essential for conservation of Succulent Karoo Biome, where indigenous invasive species like Vachellia karroo and non-native Australian acacia species are present. Furthermore, targeted management practices using effective symbionts of the studied legumes can sustain the biome's socio-economic contribution through fodder provision.
Establishing African genomics and bioinformatics programs through annual regional workshops
(Nature Research, 2024-08) Sharaf, Abdoallah; Nesengani, Lucky Tendani; Hayah, Ichrak; Kuja, Josiah Ochieng; Mdyogolo, Sinebongo; Omotoriogun, Taiwo Crossby; Odogwu, Blessing Adanta; Beedessee, Girish; Smith, Rae Marvin; Barakat, Abdelhamid; Moila, Acclaim M.; El Hamouchi, Adil; Benkahla, Alia; Boukteb, Amal; Elmouhtadi, Amine; Mafwila, Antoine Lusala; Abushady, Asmaa Mohammed; Elsherif, Assem Kadry; Ahmed, Bulbul; Wairuri, Charles; Ndiribe, Charlotte C.; Ebuzome, Chukwuike; Kinnear, Craig J.; Ndlovu, Deborah-Fay; Iraqi, Driss; El Fahime, Elmostafa; Assefa, Ermias; Ouardi, Faissal; Belharfi, Fatima Zohra; Tmimi, Fatim Zohra; Markey, Fatu Badiane; Radouani, Fouzia; Zeukeng, Francis; Mvumbi, Georges Lelo; Ganesan, Hamilton; Hanachi, Mariem; Nigussie, Helen; Charoute, Hicham; Benamri, Ichrak; Mkedder, Ikram; Haddadi, Imane; Meftah-Kadmiri, Issam; Mubiru, Jackson Franco; Domelevo Entfellner, Jean-Baka Kodjo; Rokani, Joan Bayowa; Ogwang, Joel; Daiga, Jude Bigoga; Omumbo, Judy; Ideozu, Justin Eze; Errafii, Khaoula; Labuschagne, Kim; Komi, Komi Koukoura; Tonfack, Libert Brice; Hadjeras, Lydia; Ramantswana, Madeleine; Chaisi, Mamohale; Botes, Marietjie W.; Kilian, Mariette; Kvas, Marija; Melloul, Marouane; Chaouch, Melek; Khyatti, Meriem; Abdo, Michael; Phasha-Muchemenye, Mmatshepho; Hijri, Mohamed; Mediouni, Mohammed Rida; Hassan, Mohammed Ahmed; Piro, Mohammed; Mwale, Monica; Maaloum, Mossaab; Mavhunga, Mudzuli; Olivier, Nicholas Abraham; Aminou, Oumaima; Arbani, Oumayma; Souiai, Oussema; Djocgoue, Pierre Francois; Mentag, Rachid; Zipfel, Renate Dorothea; Tata, Rolland Bantar; Megnekou, Rosette; Muzemil, Sadik; Paez, Sadye; Salifu, Samson Pandam; Kagame, Samuel Paul; Selka, Sarra; Edwards, Sean; Gaouar, Semir Bechir Suheil; Reda, Shaimaa Roshdy Abdullah; Fellahi, Siham; Khayi, Slimane; Ayed, Soumia; Madisha, Thabang; Sahil, Tulsi; Udensi, Ogbuagu Ugorji; Ras, Verena; Ezebuiro, Victor; Duru, Vincent C.; David, Xavier; Geberemichael, Yonas; Tchiechoua, Yves H.; Mungloo-Dilmohamud, Zahra; Chen, Zhiliang; Happi, Christian; Kariuki, Thomas; Ziyomo, Cathrine; Djikeng, Appolinaire; Badaoui, Bouabid; Mapholi, Ntanganedzeni; Muigai, Anne; Osuji, Julian O.; Ebenezer, ThankGod Echezona
The African BioGenome Project (AfricaBP) Open Institute for Genomics and Bioinformatics aims to overcome barriers to capacity building through its distributed African regional workshops and prioritizes the exchange of grassroots knowledge and innovation in biodiversity genomics and bioinformatics. In 2023, we implemented 28 workshops on biodiversity genomics and bioinformatics, covering 11 African countries across the 5 African geographical regions. These regional workshops trained 408 African scientists in hands-on molecular biology, genomics and bioinformatics techniques as well as the ethical, legal and social issues associated with acquiring genetic resources. Here, we discuss the implementation of transformative strategies, such as expanding the regional workshop model of AfricaBP to involve multiple countries, institutions and partners, including the proposed creation of an African digital database with sequence information relating to both biodiversity and agriculture. This will ultimately help create a critical mass of skilled genomics and bioinformatics scientists across Africa.
A revision of the family Cucurbitariaceae with additional new taxa from forest trees in Iran
(Springer, 2024-02) Eisvand, Payam; Mehrabi-Koushki, Mehdi; Crous, Pedro W.
The family Cucurbitariaceae is rich in species diversity and has a wide host range and geographic distribution. In this study, we identified 12 Cucurbitariaceae isolates which were obtained from disease symptoms in two forest trees in Khuzestan province, Iran. In addition, this family is reassessed using phylogenetic analyses based on DNA sequences from five nuclear regions (ITS, LSU, TUB2, TEF1α, and RPB2). The phylogenetic analyses showed that the present isolates represent one new genus, Nothocucurbitaria, and three new species, Allocucurbitaria galinsogisoli, Nothocucurbitaria izehica, and Parafenestella quercicola, which are described and illustrated. Furthermore, the genus Allocucurbitaria is emended to accommodate Seltsamia ulmi that grouped with the type species of Allocucurbitaria. Parafenestella pittospori and A. prunicola are recombined into the genera Neocucurbitaria and Nothocucurbitaria, respectively. Comparative analysis of single-locus trees revealed that the TUB2 and TEF1α can distinguish most genera and species in Cucurbitariaceae, while the ITS and LSU phylogenies show low resolution at both generic and species level. The best single-locus marker, RPB2, was able to distinguish all generic and most species lineages in Cucurbitariaceae.
A comprehensive molecular phylogeny of Cephalotrichum and Microascus provides novel insights into their systematics and evolutionary history
(Naturalis Biodiversity Center and Centraalbureau voor Schimmelcultures, 2024-06) Wei, T.P.; Wu, Y.M.; Zhang, X.; Zhang, H.; Crous, Pedro W.; Jiang, Y.L.
The genera Cephalotrichum and Microascus contain ecologically, morphologically and lifestyle diverse fungi in Microascaceae (Microascales, Sordariomycetes) with a world-wide distribution. Despite previous studies having elucidated that Cephalotrichum and Microascus are highly polyphyletic, the DNA phylogeny of many traditionally morphology-defined species is still poorly resolved, and a comprehensive taxonomic overview of the two genera is lacking. To resolve this issue, we integrate broad taxon sampling strategies and the most comprehensive multi-gene (ITS, LSU, tef1 and tub2) datasets to date, with fossil calibrations to address the phylogenetic relationships and divergence times among major lineages of Microascaceae. Two previously recognised main clades, Cephalotrichum (24 species) and Microascus (49 species), were re-affirmed based on our phylogenetic analyses, as well as the phylogenetic position of 15 genera within Microascaceae. In this study, we provide an up-to-date overview on the taxonomy and phylogeny of species belonging to Cephalotrichum and Microascus, as well as detailed descriptions and illustrations of 21 species of which eight are newly described. Furthermore, the divergence time estimates indicate that the crown age of Microascaceae was around 210.37 Mya (95 % HPD: 177.18–246.96 Mya) in the Late Triassic, and that Cephalotrichum and Microascus began to diversify approximately 27.07 Mya (95 % HPD: 20.47–34.37 Mya) and 70.46 Mya (95 % HPD: 56.96–86.24 Mya), respectively. Our results also demonstrate that multigene sequence data coupled with broad taxon sampling can help elucidate previously unresolved clade relationships.
Several Seiridium species (Sporocadaceae: Xylariales) cause cypress canker in South Africa
(Elsevier, 2025-02) Aylward, Janneke; Atkins, Sydney; Roets, Francois; Wingfield, Brenda D.; Wingfield, Michael J.; janneke.aylward@fabi.up.ac.za
Cypress canker is an important fungal disease caused by at least seven different Seiridium species. The disease has been known on Cupressaceae trees in South Africa since the 1980's, but its relevance was recently accentuated with an outbreak on native Widdringtonia nodiflora trees in the Western Cape. The causal agent, S. neocupressi, was previously unknown in the country, highlighting a lack of information regarding the disease in South Africa. The aim of this study was to investigate the occurrence of cypress canker and its causal agents across the country by sampling diseased Cupressaceae trees and reconsidering the identity of Seiridium strains previously collected in the country. Phylogenetic analyses revealed five known cypress canker pathogens and two putatively novel species. Seiridium cardinale was the most frequently isolated species in the Western Cape. Only two isolates of S. neocupressi were found outside the outbreak on Widdringtonia. Seiridium unicorne was most frequently isolated in the Gauteng and Free State provinces. Seiridium cancrinum, S. kenyanum and the two undescribed species were each recovered only in localised areas. Stem inoculations on x Hesperotropsis leylandii using S. cardinale, S. neocupressi and S. unicorne strains confirmed the pathogenicity of S. cardinale and S. neocupressi. In contrast, S. unicorne strains exhibited variable levels of aggressiveness. This study represents the first extensive consideration of cypress canker in South Africa and one of few studies on this important disease in the Southern Hemisphere
Production of fusel alcohols and fusel acetates by pathogenic fungi in the Ceratocystidaceae
(Elsevier, 2025-08) Mailula, Dineo M.; Wingfield, Brenda D.; Van der Nest, Magrieta Aletta; Hammerbacher, Almuth; almuth.hammerbacher@fabi.up.ac.za
The family Ceratocystidaceae includes economically important plant pathogens that vary in host preference and lifestyle. These fungi are believed to attract insect vectors, for their dispersal through their floral and fruity scents. This study aimed to identify the volatiles produced by a subset of fungi within the Ceratocystidaceae using gas chromatography coupled with mass spectrometry. The primary volatiles produced by most genera in the family were fusel alcohols and fusel acetates, but their emission rates differed significantly between genera and isolates from a single species. Ceratocystis albifundus collected from Protea cynaroides produced higher levels of fusel acetates compared to isolates from Terminalia sericea. In addition, significant differences in volatile biosynthesis were observed between isolates grown under different temperatures. Results of this study demonstrate that Ceratocystidaceae exhibit varied volatile profiles, but further research is needed to understand the ecological and physiological mechanisms underlying this plasticity.
CRISPR-Cas9 genome editing reveals that the Pgs gene of Fusarium circinatum is involved in pathogenicity, growth and sporulation
(Elsevier, 2025-03) Van Dijk, Alida; Wilson, Andi M.; Marx, Bianke; Hough, Bianca; Swalarsk-Parry, Benedicta S.; De Vos, Lieschen; Wingfield, Michael J.; Wingfield, Brenda D.; Steenkamp, Emma Theodora; alida.vandijk@up.ac.za
Fusarium circinatum, the causal agent of pine pitch canker, is one of the most destructive pathogens of Pinus species worldwide. Infections by this pathogen result in serious mortality of seedlings due to root and root collar disease, and growth reduction in trees due to canker formation and dieback. Although much is known about the population biology, genetics, and genomics of F. circinatum, relatively little is known regarding the molecular basis of pathogenicity in F. circinatum. In this study, a protoplast-based transformation using CRISPR-Cas9-mediated genome editing was utilized to functionally characterize a putative pathogenicity gene in three different strains of the fungus. In silico analyses suggested the gene likely encodes a small secreted protein, and all isolates in which it was deleted displayed significantly reduced vegetative growth and asexual spore production compared to the wild-type isolates. In pathogenicity tests, lesions induced by the deletion mutants on detached Pinus patula branches were significantly shorter than those produced by the wild-types. The putative pathogenicity gene was named Pgs reflecting its role in pathogenicity, growth, and sporulation. Future research will seek to explore the molecular mechanisms underlying the mutant phenotypes observed. Overall, this study represents a significant advance in F. circinatum research as the development and application of a Cas9-mediated gene deletion process opens new avenues for functional gene characterization underlying many of the pathogen's biological traits.
In silico characterisation of the avocado WAK/WAKL gene family with a focus on genes involved in defence against Phytophthora cinnamomi
(Frontiers Media, 2025-01) Harvey, Aaron Thomas; Van den Berg, Noelani; Swart, Velushka; velushka.swart@up.ac.za
The avocado industry faces a significant threat from the hemibiotrophic oomycete pathogen Phytophthora cinnamomi. A variably expressed defence gene during an avocado infection trial was a Wall-associated kinase (WAK). WAK and WAK-Like (WAKL) proteins are known to bind to fragmented pectin (oligogalacturonides) produced during pathogen penetration, thereby activating downstream defence-related pathways. To better understand the P. cinnamomi-avocado defence interaction, this gene family was assessed using in silico methods. In this study, previously generated RNA-sequencing data were used to associate genes with the defence response, followed by promoter- and phylogenetic analysis of these genes/proteins. The predicted proteins from these genes were modelled with AlphaFold2, and structural similarity across different rootstocks, as well as their binding affinity for oligogalacturonides, were assessed. The analysis identified 14 Persea americana (Pa)WAKs and 62 PaWAKLs across the West-Indian (pure accession reference), Dusa®, Leola™ and R0.12 avocado rootstock genomes. These genes showed distribution across the West-Indian genome’s chromosomes, with MCScanX analyses predicting tandem duplications. PaWAK/WAKL expression profiles were compared, implicating five PaWAK/WAKLs in defence. Phylogenetic and promoter analyses were conducted to predict associated defence-related pathways, focusing on stress and phytohormone-responsive pathways. Structural differences and varying oligogalacturonide binding affinities of PaWAK/WAKLs were predicted across rootstocks. These defence-related genes could be incorporated into a molecular screening tool to improve the development of resistant avocado rootstocks.
Flavonoids and anthocyanins in seagrasses : implications for climate change adaptation and resilience
(Frontiers Media, 2025-01) Botes, Jana; Ma, Xiao; Chang, Jiyang; Van de Peer, Yves; Berger, David Kenneth
Seagrasses are a paraphyletic group of marine angiosperms and retain certain adaptations from the ancestors of all embryophytes in the transition to terrestrial environments. Among these adaptations is the production of flavonoids, versatile phenylpropanoid secondary metabolites that participate in a variety of stress responses. Certain features, such as catalytic promiscuity and metabolon interactions, allow flavonoid metabolism to expand to produce novel compounds and respond to a variety of stimuli. As marine environments expose seagrasses to a unique set of stresses, these plants display interesting flavonoid profiles, the functions of which are often not completely clear. Flavonoids will likely prove to be effective and versatile agents in combating the new host of stress conditions introduced to marine environments by anthropogenic climate change, which affects marine environments differently from terrestrial ones. These new stresses include increased sulfate levels, changes in salt concentration, changes in herbivore distributions, and ocean acidification, which all involve flavonoids as stress response mechanisms, though the role of flavonoids in combatting these climate change stresses is seldom discussed directly in the literature. Flavonoids can also be used to assess the health of seagrass meadows through an interplay between flavonoid and simple phenolic levels, which may prove to be useful in monitoring the response of seagrasses to climate change. Studies focusing on the genetics of flavonoid metabolism are limited for this group, but the large chalcone synthase gene families in some species may provide an interesting topic of research. Anthocyanins are typically studied separately from other flavonoids. The phenomenon of reddening in certain seagrass species typically focuses on the importance of anthocyanins as a UV-screening mechanism, while the role of anthocyanins in cold stress is discussed less often. Both of these stress response functions would be useful for adaptation to climate change-induced deviations in tidal patterns and emersion. However, ocean warming will likely lead to a decrease in anthocyanin content, which may impact the performance of intertidal seagrasses. This review highlights the importance of flavonoids in angiosperm stress response and adaptation, examines research on flavonoids in seagrasses, and hypothesizes on the importance of flavonoids in these organisms under climate change.
Pan-genome analysis of the Enterobacter hormaechei complex highlights its genomic flexibility and pertinence as a multidrug resistant pathogen
(BioMed Central, 2025-04) De Maayer, Pieter; Green, Teigra; Jordan, Sara; Smits, Theo H.M.; Coutinho, Teresa A.
BACKGROUND : Enterobacter hormaechei is of increasing concern as both an opportunistic and nosocomial pathogen, exacerbated by its evolving multidrug resistance. However, its taxonomy remains contentious, and little is known about its pathogenesis and the broader context of its resistome. In this study, a comprehensive comparative genomic analysis was undertaken to address these issues. RESULTS : Phylogenomic analysis revealed that E. hormaechei represents a complex, comprising three predicted species, E. hormaechei, E. hoffmannii and E. xiangfangensis, with the latter putatively comprising three distinct subspecies, namely oharae, steigerwaltii and xiangfangensis. The species and subspecies all display open and distinct pan-genomes, with diversification driven by an array of mobile genetic elements including numerous plasmid replicons and prophages, integrative conjugative elements (ICE) and transposable elements. These elements have given rise to a broad, relatively conserved set of pathogenicity determinants, but also a variable set of secretion systems. The E. hormaechei complex displays a highly mutable resistome, with most taxa being multidrug resistant. CONCLUSIONS : This study addressed key issues pertaining to the taxonomy of the E. hormaechei complex, which may contribute towards more accurate identification of strains belonging to this species complex in the clinical setting. The pathogenicity determinants identified in this study could serve as a basis for a deeper understanding of E. hormaechei complex pathogenesis and virulence. The extensive nature of multidrug resistance among E. hormaechei complex strains highlights the need for responsible antibiotic stewardship to ensure effective treatment of these emerging pathogens.
Advancements in Spongospora subterranea : current knowledge, management strategies, and research gaps
(Springer, 2024-12) Strydom, R.F.; Wilson, C.R.; Tegg, R.S.; Balendres, M.A.; Van der Waals, Jacqueline Elise
Powdery scab was first documented on locally sampled potatoes in Braunschweig, Germany. A hundred and eighty-one years later, the disease has spread globally to most potato-producing regions and is considered one of the most destructive potato diseases. Here, we review the knowledge of powdery scab and causative agent, Spongospora subterranea f. sp. subterranea, highlighting research progress made in the last 7 years. Much work has been done to increase our understanding of how zoospores respond to their environment (e.g. root exudates, Ca2C signalling, and root metabolites) and the management of the disease by chemical and biological control agents. Recent research has utilised omics approaches — metabolomics, proteomics, and genomics — to gain a deeper understanding of the host–pathogen interaction in the powdery scab pathosystem. The management of powdery scab can be achieved using a combination of strategies that include (1) the planting of resistant potato varieties, (2) strategies that avoid disease (field selection and planting date), those that (3) reduce initial soil inoculum (crop rotation, organic soil amendments, and soil fumigation), and (4) in-crop approaches (soil chemical applications, biological control, proper field, fertility, crop, irrigation management, and crop sanitation). Lastly, we discuss research gaps for future research, including the disease’s interaction with other potato diseases that may be impacting disease expression and opportunities to enable a greater understanding of the powdery scab pathosystem.
Development of a molecular genetics and cell biology toolbox for the filamentous fungus Diplodia sapinea
(Public Library of Science, 2024-12-27) Oostlander, Anne Geertje; Brodde, Laura; Von Bargen, Miriam; Slippers, Bernard; Becker, Yvonne; Brandt, Ulrike; Klawonn, Frank; Grobler, Christiaan; Well, Lucas; Stenlid, Jan; Oliva, Jonas; Elfstrand, Malin; Fleissner, Andre
Diplodia sapinea (Fr.) Fuckel is a widespread fungal pathogen affecting conifers worldwide. Infections can lead to severe symptoms, such as shoot blight, canker, tree death, or blue stain in harvested wood, especially in Pinus species. Its impact on forest health is currently intensified, likely due to climate change, posing an increasing threat to global ecosystems and forestry. Despite extensive and successful research on this pathogen system, fundamental questions about its biology and plant-associated lifestyle remain unanswered. Addressing these questions will necessitate the development of additional experimental tools, including protocols for molecular genetics and cell biology approaches. In this study, we continue to address this need by establishing an Agrobacterium-mediated genetic transformation protocol for D. sapinea, enabling targeted mutagenesis and heterologous gene expression. We utilized this methodology to localize the histone H2B by tagging it with the fluorescent protein mCherry. Additionally, we established a time- and space-efficient laboratory-scale infection assay using two-week-old Pinus sylvestris seedlings. Integrating these tools in a proof-of-concept study enabled the visualization of D. sapinea in planta growth through the fluorescently labeled reporter strain.
Ecosystems managed for wildlife and livestock : contrasting herbivore impacts in a heavily grazed African savanna
(Elsevier, 2024-12) Voysey, Michael D.; Archibald, Sally; Harris, Mathew Andrew; Hempson, Gareth P.; Greve, Michelle
Replacement of diverse wild herbivore assemblages by livestock across expansive regions of African savanna grassland is predicted to modify tree-grass interactions. Yet, despite the ubiquity of this herbivore regime change, consequences of shifting from wildlife to livestock for savanna ecosystems are poorly resolved. We assessed the effects of livestock grazing on the herbaceous community of a nutrient rich, semi-arid savanna. Direct and indirect impacts - moderated via differences in tree cover - of intensive cattle grazing in communal rangelands, on the herbaceous community were contrasted with heavily utilized wild herbivore grazing systems. Communal rangelands showed a more than two-fold increase in woody cover compared to wildlife areas. Herbaceous differences between wildlife and livestock areas were driven primarily by changes in the area beneath trees, and differences in how densely wooded areas are used by wildlife and livestock. In wildlife systems, grazing intensity was much lower below than away from tree canopies, whereas in livestock systems grazing intensity was high irrespective of tree canopy presence. Away from trees, structure and functional traits of herbaceous vegetation were markedly similar between the two herbivory systems. In our study area, communal rangelands used by livestock, predominantly cattle, are not a degraded subset of wildlife areas but support near equivalent diversity, although with different grass and forb species, and warrants consideration when evaluating their contribution to biodiversity conservation in the region. Nonetheless, higher bare ground cover and the lack of tall-grass reserves mean that communally managed rangelands with low grazer diversity might be more sensitive to intra- and inter-annual climate variability since they are associated with lower forage variability.
Bacterial Isolation from natural grassland on nitrogen-free agar yields many strains without nitrogenase
(MDPI, 2025-01) Koirala, Amrit; Alshibli, Nabilah Ali; Das, Bikram K.; Brözel, Volker Siegfried
Nitrogen inputs for sustainable crop production for a growing population require the enhancement of biological nitrogen fixation. Efforts to increase biological nitrogen fixation include bioprospecting for more effective nitrogen-fixing bacteria. As bacterial nitrogenases are extremely sensitive to oxygen, most primary isolation methods rely on the use of semisolid agar or broth to limit oxygen exposure. Without physical separation, only the most competitive strains are obtained. The distance between strains provided by plating on solid media in reduced oxygen environments has been found to increase the diversity of culturable potential diazotrophic bacteria. To obtain diverse nitrogen-fixing isolates from natural grasslands, we plated soil suspensions from 27 samples onto solid nitrogen-free agar and incubated them under atmospheric and oxygen-reducing conditions. Putative nitrogen fixers were confirmed by subculturing in liquid nitrogen-free media and PCR amplification of the nifH genes. Streaking of the 432 isolates on nitrogen-rich R2A revealed many cocultures. In most cases, only one community member then grew on NFA, indicating the coexistence of nonfixers in coculture with fixers when growing under nitrogen-limited conditions. To exclude isolates able to scavenge residual nitrogen, such as that from vitamins, we used a stringent nitrogen-free medium containing only 6.42 μmol/L total nitrogen and recultured them in a nitrogen-depleted atmosphere. Surprisingly, PCR amplification of nifH using various primer pairs yielded amplicons from only 17% of the 442 isolates. The majority of the nifH PCR-negative isolates were Bacillus and Streptomyces. It is unclear whether these isolates have highly effective uptake systems or nitrogen reduction systems that are not closely aligned with known nitrogenase families. We advise caution in determining the nitrogen fixation ability of plants from growth on nitrogen-free media, even where the total nitrogen is very limited.
Effects of exogenous application of methyl jasmonate and salicylic acid on the physiological and molecular response of ‘Dusa’ avocado to Rosellinia necatrix
(American Phytopathological Society, 2024-07) Moreno-Pérez, Ana; Martínez-Ferri, Elsa; Van den Berg, Noelani; Pliego, Clara
Methyl jasmonate (MeJA) and salicylic acid (SA) are important in mediating plant responses to abiotic and biotic stresses. MeJA and SA can act as elicitors by triggering plant defense responses similar to those induced by pathogens and may even provide long-term protection against them. Thus, exogenous application of MeJA and SA could protect susceptible avocado plants against white root rot (WRR) disease caused by the necrotrophic fungus Rosellinia necatrix, one of the main diseases affecting avocado orchards. This work evaluates the effects of MeJA or SA on the physiological and molecular response of susceptible ‘Dusa’ avocado rootstock and their ability to provide some protection against WRR. The application of MeJA and SA in avocado increased photoprotective mechanisms (nonphotochemical chlorophyll fluorescence quenching) and upregulated the glutathione S-transferase, suggesting the triggering of mechanisms closely related to oxidative stress relief and reactive oxygen species scavenging. In contrast to SA, MeJA’s effects were more pronounced at the morphoanatomical level, including functional traits such as high leaf mass area, high stomatal density, and high root/shoot ratio, closely related to strategies to cope with water scarcity and WRR disease. Moreover, MeJA upregulated a greater number of defense-related genes than SA, including a glu protease inhibitor, a key gene in avocado defense against R. necatrix. The overall effects of MeJA increased ‘Dusa’ avocado tolerance to R. necatrix by inducing a primed state that delayed WRR disease symptoms. These findings point toward the use of MeJA application as an environmentally friendly strategy to mitigate the impact of this disease on susceptible avocado orchards.
Plasmids encode and can mobilize onion pathogenicity in Pantoea agglomerans
(Oxford University Press, 2025-03) Shin, Gi Yoon; Asselin, Jo Ann; Smith, Amy; Aegerter, Brenna; Coutinho, Teresa A.; Zhao, Mei; Dutta, Bhabesh; Mazzone, Jennie; Neupane, Ram; Gugino, Beth; Hoepting, Christy; Khanal, Manzeal; Malla, Subas; Nischwitz, Claudia; Sidhu, Jaspreet; Burke, Antoinette Machado; Davey, Jane; Uchanski, Mark; Derie, Michael L.; Du Toit, Lindsey J.; Stresow-Cortez, Stephen; Bonasera, Jean M.; Stodghill, Paul; Kvitko, Brian
Pantoea agglomerans is one of four Pantoea species reported in the USA to cause bacterial rot of onion bulbs. However, not all P. agglomerans strains are pathogenic to onion. We characterized onion-associated strains of P. agglomerans to elucidate the genetic and genomic signatures of onion-pathogenic P. agglomerans. We collected >300 P. agglomerans strains associated with symptomatic onion plants and bulbs from public culture collections, research laboratories, and a multi-year survey in 11 states in the USA. Combining the 87 genome assemblies with 100 high-quality, public P. agglomerans genome assemblies we identified two well-supported P. agglomerans phylogroups. Strains causing severe symptoms on onion were only identified in Phylogroup II and encoded the HiVir pantaphos biosynthetic cluster, supporting the role of HiVir as a pathogenicity factor. The P. agglomerans HiVir cluster was encoded in two distinct plasmid contexts: (i) as an accessory gene cluster on a conserved P. agglomerans plasmid (pAggl), or (ii) on a mosaic cluster of plasmids common among onion strains (pOnion). Analysis of closed genomes revealed that the pOnion plasmids harbored alt genes conferring tolerance to Allium thiosulfinate defensive chemistry and many harbored cop genes conferring resistance to copper. We demonstrated that the pOnion plasmid pCB1C can act as a natively mobilizable pathogenicity plasmid that transforms P. agglomerans Phylogroup I strains, including environmental strains, into virulent pathogens of onion. This work indicates a central role for plasmids and plasmid ecology in mediating P. agglomerans interactions with onion plants, with potential implications for onion bacterial disease management.
Self-growth suppression in Bradyrhizobium diazoefficiens is caused by a diffusible antagonist
(Oxford University Press, 2025-03) Sandhu, Armaan Kaur; Fischer, Brady R.; Subramanian, Senthil; Hoppe, Adam D.; Brözel, Volker Siegfried
Microbes in soil navigate interactions by recognizing kin, forming social groups, exhibiting antagonistic behavior, and engaging in competitive kin rivalry. Here, we investigated a novel phenomenon of self-growth suppression (sibling rivalry) observed in Bradyrhizobium diazoefficiens USDA 110. Swimming colonies of USDA 110 developed a distinct demarcation line and inter-colony zone when inoculated adjacent to each other. In addition to self, USDA 110 suppressed growth of other Bradyrhizobium strains and several other soil bacteria. We demonstrated that the phenomenon of sibling rivalry is due to growth suppression but not cell death. The cells in the inter-colony zone were culturable but had reduced respiratory activity, ATP levels, and motility. The observed growth suppression was due to the presence of a diffusible effector compound. This effector was labile, preventing extraction, and identification, but it is unlikely a protein or a strong acid or base. This counterintuitive phenomenon of self-growth suppression suggests a strategic adaptation for conserving energy and resources in competitive soil environments. Bradyrhizobium’s utilization of antagonism including self-growth suppression likely provides a competitive advantage for long-term success in soil ecosystems.
Bradyrhizobium diazoefficiens cultures display phenotypic heterogeneity
(Oxford University Press, 2025-03) Sarao, Sukhvir K.; Sandhu, Armaan K.; Hanson, Ryan L.; Govil, Tanvi; Brözel, Volker Siegfried
Bacteria growing in liquid culture are assumed to be homogenous in phenotype. Characterization of individual cells shows that some clonal cultures contain more than one phenotype. Bacteria appear to employ bet hedging where various phenotypes help the species survive in diverse niches in soil and rhizosphere environments. We asked whether the agriculturally significant bacterium Bradyrhizobium diazoefficiens USDA 110, which fixes nitrogen with soybean plants, displays phenotypic heterogeneity when grown under laboratory conditions. We observed differential binding of sugar-specific lectins in isogenic populations, revealing differential surface properties. We employed Percoll™ density gradient centrifugation to separate clonal populations of exponential and stationary phase B. diazoefficiens into four fractions and characterized their phenotype by proteomics. Specific phenotypes were then characterized in detail. Fractions varied by cell size, polyhydroxyalkanoate content, lectin binding profile, growth rate, cellular adenosine triphosphate, chemotaxis, and respiration activity. Phenotypes were not heritable because the specific buoyant densities of fractions equilibrated within 10 generations. We propose that heterogeneity helps slow growing B. diazoefficiens proliferate and maintain populations in the different environments in soil and the rhizosphere.

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