The soil-borne white root rot pathogen Rosellinia necatrix expresses antimicrobial proteins during host colonization.

Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited availability of genomic resources for R. necatrix has complicated a thorough understanding of its infection biology. Here, we sequenced nine R. necatrix strains with Oxford Nanopore sequencing technology, and with DNA proximity ligation we generated a gapless assembly of one of the genomes into ten chromosomes. Whereas many filamentous pathogens display a so-called two-speed genome with more dynamic and more conserved compartments, the R. necatrix genome does not display such genome compartmentalization. It has recently been proposed that fungal plant pathogens may employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection. In the predicted secretome of R. necatrix, 26 putative antimicrobial effector proteins were identified, nine of which are expressed during plant colonization. Two of the candidates were tested, both of which were found to possess selective antimicrobial activity. Intriguingly, some of the inhibited bacteria are antagonists of R. necatrix growth in vitro and can alleviate R. necatrix infection on cotton plants. Collectively, our data show that R. necatrix encodes antimicrobials that are expressed during host colonization and that may contribute to modulation of host-associated microbiota to stimulate disease development.

Epigenetic regulation of nuclear processes in fungal plant pathogens.

Through the association of protein complexes to DNA, the eukaryotic nuclear genome is broadly organized into open euchromatin that is accessible for enzymes acting on DNA and condensed heterochromatin that is inaccessible. Chemical and physical alterations to chromatin may impact its organization and functionality and are therefore important regulators of nuclear processes. Studies in various fungal plant pathogens have uncovered an association between chromatin organization and expression of in planta-induced genes that are important for pathogenicity. This review discusses chromatin-based regulation mechanisms as determined in the fungal plant pathogen Verticillium dahliae and relates the importance of epigenetic transcriptional regulation and other nuclear processes more broadly in fungal plant pathogens.

Oomycete metabolism is highly dynamic and reflects lifestyle adaptations.

The selective pressure of pathogen-host symbiosis drives adaptations. How these interactions shape the metabolism of pathogens is largely unknown. Here, we use comparative genomics to systematically analyse the metabolic networks of oomycetes, a diverse group of eukaryotes that includes saprotrophs as well as pathogens of animal- and plant pathogens, the latter causing devastating diseases with significant economic and/or ecological impact. In our analyses of 44 oomycete species, we uncover considerable variation in metabolism that can be linked to lifestyle differences. Comparisons of metabolic gene content reveal that plant pathogenic oomycetes have a bipartite metabolism consisting of a conserved core and an accessory set. The accessory set can be associated with the degradation of defence compounds produced by plants when challenged by pathogens. Obligate biotrophic oomycetes have smaller metabolic networks, and taxonomically distantly related biotrophic lineages display convergent evolution by repeated gene losses in both the conserved as well as the accessory set of metabolism. When investigating to what extent the metabolic networks in obligate biotrophs differ from those in hemibiotrophic plant pathogens, we observe that the losses of metabolic enzymes in obligate biotrophs are not random and that gene losses predominantly influence the terminal branches of the metabolic networks. Our analyses represent the first metabolism-focused comparison of oomycetes at this scale and will contribute to a better understanding of the evolution of oomycete metabolism in relation to lifestyle adaptation.

Genetic diversity of the banana Fusarium wilt pathogen in Cuba and across Latin America and the Caribbean.

Fusarium wilt of bananas (FWB) is a severe plant disease that leads to substantial losses in banana production worldwide. It remains a major concern for Cuban banana cultivation. The disease is caused by members of the soil-borne Fusarium oxysporum species complex. However, the genetic diversity among Fusarium species infecting bananas in Cuba has remained largely unexplored. In our comprehensive survey, we examined symptomatic banana plants across all production zones in the country, collecting 170 Fusarium isolates. Leveraging genotyping-by-sequencing and whole-genome comparisons, we investigated the genetic diversity within these isolates and compared it with a global Fusarium panel. Notably, typical FWB symptoms were observed in Bluggoe cooking bananas and Pisang Awak subgroups across 14 provinces. Our phylogenetic analysis revealed that F. purpurascens, F. phialophorum, and F. tardichlamydosporum are responsible for FWB in Cuba, with F. tardichlamydosporum dominating the population. Furthermore, we identified between five and seven distinct genetic clusters, with F. tardichlamydosporum isolates forming at least two subgroups. This finding underscores the high genetic diversity of Fusarium spp. contributing to FWB in the Americas. Our study sheds light on the population genetic structure and diversity of the FWB pathogen in Cuba and the broader Latin American and Caribbean regions.

Segmental duplications drive the evolution of accessory regions in a major crop pathogen.

Many pathogens evolved compartmentalized genomes with conserved core and variable accessory regions (ARs) that carry effector genes mediating virulence. The fungal plant pathogen Fusarium oxysporum has such ARs, often spanning entire chromosomes. The presence of specific ARs influences the host range, and horizontal transfer of ARs can modify the pathogenicity of the receiving strain. However, how these ARs evolve in strains that infect the same host remains largely unknown. We defined the pan-genome of 69 diverse F. oxysporum strains that cause Fusarium wilt of banana, a significant constraint to global banana production, and analyzed the diversity and evolution of the ARs. Accessory regions in F. oxysporum strains infecting the same banana cultivar are highly diverse, and we could not identify any shared genomic regions and in planta-induced effectors. We demonstrate that segmental duplications drive the evolution of ARs. Furthermore, we show that recent segmental duplications specifically in accessory chromosomes cause the expansion of ARs in F. oxysporum. Taken together, we conclude that extensive recent duplications drive the evolution of ARs in F. oxysporum, which contribute to the evolution of virulence.

Characterization of the Ligand Properties of Donor-stabilized Pnictogenyltrielanes.

A general synthesis and the characterization of novel alkyl-substituted NHC-stabilized pnictogenylboranes NHC ⋅ BH2 ER2 (NHC=N-heterocyclic carbene, E=P, As; R2 =Me2 , Ph2 , t BuH, Cy2 , (SiMe3 )2 ) are reported. These compounds were reacted with Ni(CO)4 to the corresponding complexes of the type [(NHC ⋅ BH2 ER2 )Ni(CO)3 ] to determine their donor strength by Tolman Electronic Parameters (TEPs) and their steric demand as ligands compared to classical phosphines, superbasic phosphines and other commonly applied donor systems. The results show that the NHC-stabilized pnictogenyltrielanes can be considered as being highly basic, while their steric influence depends strongly on the organic residues as well as the donor attached to the {BH2 } moiety. Although weaker than commonly used superbasic phosphines, the donor strength of pnictogenyltrielanes in general can be classified as of similar strength as NHCs. The steric and electronic properties can easily be modified by alkyl substitution as evident from the TEP trends.

NHC-Stabilized Mixed Group 13/14/15 Element Hydrides.

The syntheses of novel N-heterocyclic carbene (NHC) adducts of group 13, 14 and 15 element hydrides are reported. Salt metathesis reactions between NaPH2 and IDipp ⋅ GeH2 BH2 OTf (1) (IDipp=1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene) led to mixtures of the two isomers IDipp ⋅ GeH2 BH2 PH2 (2 a) and IDipp ⋅ BH2 GeH2 PH2 (2 b); by altering the reaction conditions an almost exclusive formation of 2 b was achieved. Attempts to purify mixtures of 2 a and 2 b by re-crystallization from THF afforded a salt [IDipp ⋅ GeH2 BH2 ⋅ IDipp][PHGeH2 BH2 PH2 BH2 GeH2 ] (4) that contains the novel anionic cyclohexyl-like inorganic heterocycle [PHGeH2 BH2 PH2 BH2 GeH2 ]- . In addition, the borane adducts IDipp ⋅ GeH2 BH2 PH2 BH3 (3 a) and IDipp ⋅ BH2 GeH2 PH2 BH3 (3 b) as even longer chain compounds were obtained from reactions of 2 a/2 b with H3 B ⋅ SMe2 and were studied by NMR spectroscopy. Accompanying DFT computations give insight into the mechanism and energetics associated with 2 a/2 b isomerization as well as their decomposition pathways.

Møller-Plesset Adiabatic Connection at Large Coupling Strengths for Open-Shell Systems.

We study the adiabatic connection that has as weak-coupling expansion the Møller-Plesset perturbation series, generalizing to the open-shell case previous closed-shell results for the large-coupling limit. We first focus on the hydrogen atom with fractional spins, providing results along the adiabatic connection from small to large coupling strengths. We reveal an intriguing phase diagram and an equation for the large-coupling leading order that has closed-form solutions for specific choices of its relevant quantum numbers. We then show that the hydrogen atom results provide variational estimates for the large-coupling leading terms for the general many-electron open-shell case in terms of functionals of the Hartree-Fock α-spin and ß-spin densities.

An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation.

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial coinhabitants. Also, plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne, fungal, xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life stage-specific manner and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota.

Detection and quantification of the histone code in the fungal genus Aspergillus.

In eukaryotes, the combination of different histone post-translational modifications (PTMs) - the histone code - impacts the chromatin organization as compact and transcriptionally silent heterochromatin or accessible and transcriptionally active euchromatin. Although specific histone PTMs have been studied in fungi, an overview of histone PTMs and their relative abundance is still lacking. Here, we used mass spectrometry to detect and quantify histone PTMs in three fungal species belonging to three distinct taxonomic sections of the genus Aspergillus (Aspergillus niger, Aspergillus nidulans (two strains), and Aspergillus fumigatus). We overall detected 23 different histone PTMs, including a majority of lysine methylations and acetylations, and 23 co-occurrence patterns of multiple histone PTMs. Among those, we report for the first time the detection of H3K79me1, H3K79me2, and H4K31ac in Aspergilli. Although all three species harbour the same PTMs, we found significant differences in the relative abundance of H3K9me1/2/3, H3K14ac, H3K36me1 and H3K79me1, as well as the co-occurrence of acetylation on both K18 and K23 of histone H3 in a strain-specific manner. Our results provide novel insights about the underexplored complexity of the histone code in filamentous fungi, and its functional implications on genome architecture and gene regulation.

A highly polymorphic effector protein promotes fungal virulence through suppression of plant-associated Actinobacteria.

Plant pathogens secrete effector proteins to support host colonization through a wide range of molecular mechanisms, while plant immune systems evolved receptors to recognize effectors or their activities to mount immune responses to halt pathogens. Importantly, plants do not act as single organisms, but rather as holobionts that actively shape their microbiota as a determinant of health. The soil-borne fungal pathogen Verticillium dahliae was recently demonstrated to exploit the VdAve1 effector to manipulate the host microbiota to promote vascular wilt disease in the absence of the corresponding immune receptor Ve1. We identify a multiallelic V. dahliae gene displaying c. 65% sequence similarity to VdAve1, named VdAve1-like (VdAve1L), which shows extreme sequence variation, including alleles that encode dysfunctional proteins, indicative of selection pressure to overcome host recognition. We show that the orphan cell surface receptor Ve2, encoded at the Ve locus, does not recognize VdAve1L. Additionally, we demonstrate that the full-length variant VdAve1L2 possesses antimicrobial activity, like VdAve1, yet with a divergent activity spectrum, that is exploited by V. dahliae to mediate tomato colonization through the direct suppression of antagonistic Actinobacteria in the host microbiota. Our findings open up strategies for more targeted biocontrol against microbial plant pathogens.

A General Pathway towards NHC ⋠GaH2 (OTf) Adducts - The Key for the Synthesis of NHC-Stabilized Cationic 13/15 Chain Compounds of Gallium.

A general pathway towards NHC (NHC=N-heterocyclic carbene)-stabilized galliummonotriflates NHC ⋅ GaH2 (OTf) (NHC=IDipp, 1 a; IPr2 Me2 , 1 b; IMes, 1 c; IDipp=1,3-bis(2,6-diisopropylphenyl)-imidazolin-2-ylidene, IPr2 Me2 =1,3-bis-(diisopropyl)-4,5-dimethyl-imidazolin-2-ylidene, IMes=1,3-bis(2,4,6-trimethylphenyl)-imidazolin-2-ylidene) is reported. Quantum chemical calculations give detailed insight into the underlying reaction pathway. The obtained NHC ⋅ GaH2 (OTf) compounds were employed in reactions with donor-stabilized pnictogenylboranes to synthesize the elusive cationic parent 13/15/13 chain compounds [IDipp ⋅ GaH2 ER2 E'H2 ⋅ D][OTf] (3 a: D=IDipp, E=P, E'=B, R=H; 3 b: D=NMe3 , E=P, E'=B, R=H, 3 c: D=NMe3 , E=P, E'=B, R=Ph, 3 d: D=IDipp, E=P, E'=Ga, R=H). Supporting computational studies highlight the electronic features of the products.

Reactivity of [Cp''2 Zr(η1:1 -E4 )] (E=P, As) towards Nucleophiles.

The functionalization of the polypnictogen ligand complexes [Cp''2 Zr(η1:1 -E4 )] (E=P (1 a), As (1 b); Cp''=1,3-di-tertbutyl-cyclopentadienyl) is focused to modify the features of the polypnictogen unit to explore new synthetic pathways for further transformations. The reaction behavior of 1 towards main group nucleophiles is investigated. The reaction of 1 a with t BuLi leads to the ionic product Li[Cp''2 Zr(η1:1 -P4 t Bu)] (2) where an organic group is attached to a bridgehead phosphorus atom of the butterfly unit. Further reactions of 2 with quenching electrophilic reagents enable the introduction of other substituents. Moreover, a condensation of 2 to [(Cp''2 Zr)2 (µ,η1:1:1:1 -P8 t Bu2 )] (3), containing a novel P8 -unit, has been observed. The reaction of 1 with LiNMe2 and LiCH2 SiMe3 leads to a partial fragmentation of the E4 unit and the compounds [Cp''2 Zr(η2 -E3 Nu)] (Nu=NMe2 : E=P (6 a), As (6 b); Nu=CH2 SiMe3 : E=P (7 a), As (7 b)) are formed.

Transformation of the cyclo-P5 Middle Deck in [(Cp*Fe)(Cp'''Co)(µ,η5 :η4 -P5 )] upon Functionalization - A Comprehensive Study of Reactivity.

The heterobimetallic triple-decker complex [(Cp*Fe)(Cp'''Co)(µ,η5 : η4 -P5 )] (1) was functionalized by main group nucleophiles and subsequently electrophilically quenched or oxidized. Reacting 1 with group 14 nucleophiles revealed different organo-substituted P5 R middle-decks depending on the steric and electronic effects of the used alkali metal organyls (2: R=tBu; 3: R=Me). Further, with group 15 nucleophiles, the first structural characterized monosubstituted complexes with phosphanides could be obtained as P5 ligands containing exocyclic {PR2 } units (4: R=Cy, H; 5: R=Ph). These monoanionic complexes 2-5 were isolated and subsequent electrophilic quenching revealed novel types of neutral functionalized polyphosphorus complexes. These complexes bear formal chains of P5 R'R'' (6: R'=tBu, R'=Me) in a 1,3-disubstitution pattern or P6 R'R''R''' units (7: R'=Cy, R''=H, R'''=Me; 8: R'=Me, R''=Ph, R'''=Me) in a 1,1,3-substitution as middle-decks stabilized by one {Cp'''Co} and one {Cp*Fe} fragment. One-electron oxidation of 2, 3 or 5 by AgBF4 gave access to paramagnetic triple-decker complexes bearing P5 R middle-decks in various coordination fashions (R=tBu (10), R=PPh2 (12)). Interestingly, for R=Me (11), a dimerization is observed revealing a diamagnetic tetranuclear cluster containing a unique dihydrofulvalene-type P10 R2 ligand. All complexes were characterized by crystallographic and spectroscopic methods (EPR, multinuclear NMR and mass spectrometry) and their electronic structures were elucidated by DFT calculations.

Organometallic-Organic Hybrid Assemblies Featuring the Diantimony Complex [Cp2 Mo2 (CO)4 (µ,η2 -Sb2 )], AgI Ions and N-Donor Molecules as Building Blocks.

The reactions of the organometallic ligand complex [Cp2 Mo2 (CO)4 (µ,η2 -Sb2 )] (C) with Ag[TEF] ([TEF]- =[Al{OC(CF3 )3 }4 ]- ) in the presence of a number of di- or polytopic N-donor molecules (1,6,7,12-tetraazaperylene (L1), 2,2'-bipyrimidine (L2), 4,4'-bipyridine (L3), trans-1,2-di(4-pyridyl)ethylene (L4) and 1,3-di(4-pyridyl)propane (L5)), were studied. Depending on the reaction stoichiometry and choice of linker, these reactions lead to the selective formation of dimeric or tetrameric supramolecular coordination complexes as well as 1D and 2D coordination polymers (CPs). The presented compounds are unique examples of supramolecular complexes incorporating both organometallic Sb-donor and organic N-donor molecules as ligands to stabilize metal ions. Moreover, one of the formed compounds, the CP [Ag4 (η2 : 1 -C)4 (L4)4 ]n [TEF]4n , represents an exceptional 1D polymer incorporating both N- and Sb-donor ligands as connectors for metal ions.

Coordination of Pnictogenylboranes Towards Tl(I) Salts and a Tl- Mediated P-P Coupling.

The coordination chemistry of only Lewis-base (LB)-stabilized pnictogenylboranes EH2 BH2 ⋅NMe3 (E=P, As) towards Tl(I) salts has been studied. The reaction of Tl[BArCl ] (BArCl =[B(3,5-C6 H3 Cl2 )4 ]- ) with the corresponding pnictogenylborane results in the formation of [Tl(EH2 BH2 ⋅NMe3 )][BArCl ] (1 a: E=P; 1 b: E=As). Whereas the Tl ion in 1 a/b is monocoordinated, the exchange of the weakly coordinating anion (WCA) in the Tl(I) salt leads to the formation of a trigonal pyramidal coordination mode at the Tl atom by coordination of three equivalents of EH2 BH2 ⋅ NMe3 in [Tl(EH2 BH2 ⋅ NMe3 )3 ][WCA] (2 a: E=P, WCA=TEFCl ; 2 b: E=As, WCA=TEF) (TEF=[Al{OC(CF3 )3 }4 ]- , TEFCl =[Al{(OC(CF3 )2 (CCl3 )}4 ]- ). Furthermore, by using two equivalents of PH2 BH2 ⋅NMe3 , a Tl(I)-mediated P-P coupling takes place in CH2 Cl2 as solvent resulting in [Me3 N⋅BH2 PH2 PHBH2 ⋅NMe3 ][WCA] (WCA=TEF, 3 a; BArCl , 3 b; TEFCl , 3 c). In contrast, for the arsenic derivatives 1 b and 2 b, no coupling reaction is observed. The underlying chemical processes are elucidated by quantum chemical computations.

NHC-Stabilised Parent Tripentelyltrielanes.

A missing family of the extremely air sensitive tripentelyltrielanes was discovered. Their stabilisation was achieved by using the bulky NHC IDipp (NHC=N-heterocyclic carbene, IDipp=1,3-bis(2,6-diisopropylphenyl)-imidazolin-2-ylidene). The tripentelylgallanes and tripentelylalanes IDipp ⋅ Ga(PH2 )3 (1 a), IDipp ⋅ Ga(AsH2 )3 (1 b), IDipp ⋅ Al(PH2 )3 (2 a) and IDipp ⋅ Al(AsH2 )3 (2 b) were synthesised by salt metathesis of IDipp ⋅ ECl3 (E=Al, Ga, In) with alkali metal pnictogenides such as NaPH2 /LiPH2 ⋅ DME and KAsH2 , respectively. Moreover, the detection of the first NHC-stabilised tripentelylindiumane IDipp ⋅ In(PH2 )3 (3) was possible by multinuclear NMR spectroscopy. Initial investigations of the coordination ability of these compounds resulted in the successful isolation of the coordination compound [IDipp ⋅ Ga(PH2 )2 (µ3 -PH2 {HgC6 F4 }3 )] (4) by reaction of 1 a with (HgC6 F4 )3 . The compounds were characterised by multinuclear NMR spectroscopy as well as single crystal X-ray diffraction studies. Supporting computational studies highlight the electronic features of the products.

Three- and Five-Membered Anionic Chains of Pnictogenylboranes.

An unprecedented family of three- and five-membered substituted anionic derivatives of parent pnictogenylboranes is herein reported. Reacting various combinations of the pnictogenylboranes H2 E'-BH2 -NMe3 (E'=P, As) with pnictogen-based nucleophiles MER1R2 (E=P, As; R1=H, R2=t Bu; R1=R2=Ph; M=Na, K) allows for the isolation of the unsymmetrical products [Na(18-crown-6)][H2 E'-BH2 -EHt Bu] (3: E=E'=P; 4: E=E'=As; 5: E=As, E'=P) and [M(C)][H2 E'-BH2 -EPh2 ] (7: E=E'=P, M=Na, C=18-crown-6; 8: E=E'=As; M=K, C=[2.2.2]cryptand; 9: E=P, E'=As, M=Na, C=[2.2.2]cryptand; 10: E=As, E'=P, M=K, C=[2.2.2]cryptand). [Na(18-crown-6)][H2 As-BH2 -t BuPH-BH3 ] (6) is only accessible by a different pathway, using t BuPH2 , BH3 ⋅ SMe2 and NaNH2 as starting materials. Additionally, the synthesis of symmetrical diphenyl-substituted compounds [M(18-crown-6)][Ph2 E-BH2 -EPh2 ] (11: E=P, M=Na; 12: E=As, M=K) is reported which can be regarded as isostructural inorganic, negatively charged analogs of dppm (1,1-bis(diphenylphosphino)methane) and dpam (1,1-bis(diphenylarsino)methane). Furthermore, an elongation of the pnictogen boron backbone in compounds 3, 7 and 9' (similar compound to 9, stabilized however by 18-crown-6), is attainable by reacting them with the pnictogenylboranes H2 E'-BH2 -NMe3 leading to corresponding five-membered chain-like compounds [Na(18-crown-6)][H2 E-BH2 -R1R2P-BH2 -E'H2 ] (E=E'=P, R1=H, R2=t Bu (13); E=E'=P, R1=R2=Ph (14); E=E'=As, R1=R2=Ph (15); E=P, E'=As, R1=R2=Ph (16)). Finally, the thermodynamics of the reaction pathways were evaluated by quantum chemical computations.

Large-Z atoms in the strong-interaction limit of DFT: Implications for gradient expansions and for the Lieb-Oxford bound.

We numerically study the strong-interaction limit of the exchange-correlation functional for neutral atoms and Bohr atoms as the number of electrons increases. Using a compact representation, we analyze the second-order gradient expansion, comparing it with the one for exchange (weak interaction limit). The two gradient expansions, at strong and weak interaction, turn out to be very similar in magnitude but with opposite signs. We find that the point-charge plus continuum model is surprisingly accurate for the gradient expansion coefficient at strong coupling, while generalized gradient approximations, such as Perdew-Burke-Ernzerhof (PBE) and PBEsol, severely underestimate it. We then use our results to analyze the Lieb-Oxford bound from the point of view of slowly varying densities, clarifying some aspects on the bound at a fixed number of electrons.

Dissemination of Fusarium Wilt of Banana in Mozambique Caused by Fusarium odoratissimum Tropical Race 4.

Fusarium wilt of banana (FWB) is a serious soil-borne fungal disease. In the previous century, FWB already destroyed Gros Michel-based banana cultures in Central America, and currently, the disease threatens all major banana-producing regions of the world. The causal agents of these epidemics, however, are diverse. Gros Michel was infected by a wide range of Fusarium species, the so-called Race 1 strains, whereas the contemporary Cavendish-based cultures are affected by Fusarium odoratissimum, colloquially called Tropical Race 4 (TR4). TR4 was reported in Mozambique on two commercial banana farms in 2013, but no incursions were found outside the farm boundaries in 2015, suggesting that the disease was under control. Here we report the presence of TR4 outside of these farm boundaries. We obtained fungal samples from 13 banana plants in smallholder and roadside plantings at various locations throughout northern Mozambique. These samples tested positive for TR4 by molecular diagnostics and in greenhouse pathogenicity assays. The results were confirmed with reisolations, thereby completing Koch's postulates. To study the diversity of TR4 isolates in Mozambique, we selected five samples for whole-genome sequencing. Comparison with a global collection of TR4 samples revealed very little genetic variation, indicating that the fungus is clonally spreading in Mozambique. Furthermore, isolates from Mozambique are clearly genetically separated from other geographic incursions, and thus we cannot trace the origin of TR4 in Mozambique. Nevertheless, our data demonstrates the dissemination of TR4 in Mozambique, underscoring the failure of disease management strategies. This threatens African banana production.