Differential Pathogenicity and Secreted in Xylem Gene Patterns in Fusarium spp. Causing Onion Basal Rot.

It is hypothesized that the virulence of phytopathogenic fungi is mediated through the secretion of small effector proteins that interfere with the defence responses of the host plant. In Fusarium oxysporum, one family of effectors, the Secreted In Xylem (SIX) genes, has been identified. We sought to characterize the diversity and evolution of the SIX genes in the banana-infecting lineages of F. oxysporum f. sp. cubense (Foc). Whole-genome sequencing data were generated for the 23 genetic lineages of Foc, which were subsequently queried for the 14 known SIX genes (SIX1-SIX14). The sequences of the identified SIX genes were confirmed in a larger collection of Foc isolates. Genealogies were generated for each of the SIX genes identified in Foc to further investigate the evolution of the SIX genes in Foc. Within Foc, variation of the SIX gene profile, including the presence of specific SIX homologues, correlated with the pathogenic race structure of Foc. Furthermore, the topologies of the SIX gene trees were discordant with the topology of an infraspecies phylogeny inferred from EF-1α/RPB1/RPB2 (translation elongation factor-1α/RNA polymerase II subunit I/RNA polymerase II subunit II). A series of topological constraint models provided strong evidence for the horizontal transmission of SIX genes in Foc. The horizontal inheritance of pathogenicity genes in Foc counters previous assumptions that convergent evolution has driven the polyphyletic phylogeny of Foc. This work has significant implications for the management of Foc, including the improvement of diagnostics and breeding programmes.

Members of the Fusarium oxysporum species complex include pathogenic and non-pathogenic isolates and infect a broad range of plant species. F. oxysporum f. sp. cubense (Foc) causes the destructive Fusarium wilt of banana, and the recently emerged Foc tropical race 4 strain threatens the global banana industry. Secreted in xylem (SIX) genes encode for F. oxysporum effector proteins that are associated with virulence in pathogenic F. oxysporum, however they have rarely been reported from non-pathogenic F. oxysporum isolates. Our recent survey of asymptomatic banana plants grown in Foc-infested fields in Queensland and northern NSW revealed that diverse Fusarium spp, including F. oxysporum, reside in the plant roots and pseudostem without causing obvious damage to the plant. Intriguingly, we amplified SIX genes from several of the putative endophytic F. oxysporum isolates identified in the survey and found that they differ in their profile to known Foc SIX genes. To study the role of the endophytic F. oxysporum isolates in planta and the biological function of their SIX genes in more detail, we will re-inoculate cultivated and wild diploid banana lines with the endophytic F. oxysporum strains under glasshouse conditions to assess if they are non-pathogenic on banana. Secondly, we will determine whether the endophytic F. oxysporum SIX genes are expressed in planta and/or in vitro and look at the transcriptome changes occurring in the host following infection. Finally, endophytic F. oxysporum strains transformed with GFP will be used to investigate the extent of fungal colonisation in the plant.

Fusarium oxysporum is a soilborne fungal plant pathogen responsible for causing disease in many economically important crops with “special forms” (formae speciales) adapted to infect specific plant hosts. F. oxysporum f. sp. pisi (FOP) is the causal agent of Fusarium wilt disease of pea. It has been reported in every country where peas are grown commercially. Disease is generally controlled using resistant cultivars possessing single major gene resistance and therefore there is a constant risk of breakdown. The main aim of this work was to characterise F. oxysporum isolates collected from diseased peas in the United Kingdom as well as FOP isolates obtained from other researchers representing different races through sequencing of a housekeeping gene and the presence of Secreted In Xylem (SIX) genes, which have previously been associated with pathogenicity in other F. oxysporum f. spp. F. oxysporum isolates from diseased United Kingdom pea plants possessed none or just one or two known SIX genes with no consistent pattern of presence/absence, leading to the conclusion that they were foot-rot causing isolates rather than FOP. In contrast, FOP isolates had different complements of SIX genes with all those identified as race 1 containing SIX1, SIX6, SIX7, SIX9, SIX10, SIX11, SIX12, and SIX14. FOP isolates that were identified as belonging to race 2 through testing on differential pea cultivars, contained either SIX1, SIX6, SIX9, SIX13, SIX14 or SIX1, SIX6, SIX13. Significant upregulation of SIX genes was also observed in planta over the early stages of infection by different FOP races in pea roots. Race specific SIX gene profiling may therefore provide potential targets for molecular identification of FOP races but further research is needed to determine whether variation in complement of SIX genes in FOP race 2 isolates results in differences in virulence across a broader set of pea differential cultivars.

Sequencing of individual chromosomes of plant pathogenic Fusarium oxysporum

Fusarium oxysporum is a globally distributed soilborne fungal pathogen causing root rots, bulb rots, crown rots and vascular wilts on a range of horticultural plants. Pathogenic F. oxysporum isolates are highly host specific and are classified as formae speciales. Narcissus is an important ornamental crop and both the quality and yield of flowers and bulbs can be severely affected by a basal rot caused by F. oxysporum f. sp. narcissi (FON); 154 Fusarium isolates were obtained from different locations and Narcissus cultivars in the United Kingdom, representing a valuable resource. A subset of 30 F. oxysporum isolates were all found to be pathogenic and were therefore identified as FON. Molecular characterisation of isolates through sequencing of three housekeeping genes, suggested a monophyletic origin with little divergence. PCR detection of 14 Secreted in Xylem (SIX) genes, previously shown to be associated with pathogenicity in other F. oxysporum f. spp., revealed different complements of SIX7, SIX9, SIX10, SIX12 and SIX13 within FON isolates which may suggest a race structure. SIX gene sequences were unique to FON and SIX10 was present in all isolates, allowing for molecular identification of FON for the first time. The genome of a highly pathogenic isolate was sequenced and lineage specific (LS) regions identified which harboured putative effectors including the SIX genes. Real-time RT-PCR, showed that SIX genes and selected putative effectors were expressed in planta with many significantly upregulated during infection. This is the first study to characterise molecular variation in FON and provide an analysis of the FON genome. Identification of expressed genes potentially associated with virulence provides the basis for future functional studies and new targets for molecular diagnostics.

Saffron corm rot (SCR), the most serious disease affecting saffron, has been confirmed to be caused by Fusarium oxysporum in previous studies. Compared to other fungal species, F. oxysporum exhibits host specialization, a special phenomenon associated with the secreted in xylem (SIX) genes. This study examined the pathogenicity specialization of F. oxysporum isolated from saffron corms with SCR disease. The results showed that this F. oxysporum strain was strongly pathogenic to saffron corms, causing SCR; weakly pathogenic to the corms of freesia, which is in the Iridaceae family along with saffron; and not pathogenic to watermelon, melon, and tomato. Other formae speciales of F. oxysporum were not pathogenic to saffron corms. This suggests that F. oxysporum saffron strains exhibit obvious pathogenicity specialization for Iridaceae spp. Subsequently, the F. oxysporum saffron strain (XHH35) genome was sequenced, and a comparative genomics study of XHH35 and three other formae speciales was conducted using OrthoVenn3. XHH35 contained 90 specific genes absent in the other three formae speciales. These genes are involved in certain key biological processes and molecular functions. Based on BLAST homology searching, the F. oxysporum saffron strain (XHH35) genome was predicted to contain seven SIX genes (SIX 4, SIX 6, SIX 7, SIX 10, SIX 11, SIX 12, and SIX 14) highly homologous to F. oxysporum f. sp. lycopersici, which was verified using polymerase chain reaction (PCR) amplification. The corresponding individual phylogenetic tree indicated that the F. oxysporum saffron strain (XHH35) showed a separate branch with different formae speciales. This study is the first-ever report of F. oxysporum f. sp. crocus, a new forma specialis. Based on the specificity of its SIX genes, the SIX 10 gene was selected to further establish a rapid identification technique for F. oxysporum f. sp. crocus, which will be useful in future research.

During the infection of a host, plant pathogenic fungi secrete small proteins called effectors, which then modulate the defence response of the host. In the Fusarium oxysporum species complex (FOSC), the secreted in xylem (SIX) gene effectors are important for host-specific pathogenicity, and are also useful markers for identifying the various host-specific lineages. While the presence and diversity of the SIX genes has been explored in many of the pathogenic lineages of F. oxysporum, there is a limited understanding of these genes in non-pathogenic, endophytic isolates of F. oxysporum. In this study, universal primers for each of the known SIX genes are designed and used to screen a panel of endophytically-associated Fusarium species isolated from healthy, asymptomatic banana tissue. SIX gene orthologues are identified in the majority of the Fusarium isolates screened in this study. Furthermore, the SIX gene profiles of these endophytic isolates do not overlap with the SIX genes present in the pathogenic lineages of F. oxysporum that are assessed in this study. SIX gene orthologues have not been commonly identified in Fusarium species outside of the FOSC nor in non-pathogenic isolates of F. oxysporum. The results of this study indicate that the SIX gene effectors may be more broadly distributed throughout the Fusarium genus than previously thought. This has important implications for understanding the evolution of pathogenicity in the FOSC.

Secreted in Xylem (SIX) are small effector proteins released by Fusarium oxysporum f.sp. cubense (Foc) into the plant's xylem sap disrupting the host's defence responses causing Fusarium wilt disease resulting in a significant decline in banana crop yields and economic losses. Notably, different races of Foc possess unique sets of SIX genes responsible for their virulence, however, these genes remain underutilized, despite their potential as biomarkers for early disease detection. Herein, we identified seven SIX genes i.e. SIX1, SIX2, SIX4, SIX6, SIX8a, SIX9a and SIX13 present in Foc Tropical Race 4 (FocTR4), while only SIX9b in Foc Race 1 (Foc1). Analysis of SIX gene expression in infected banana roots revealed differential patterns during infection providing valuable insights into host-pathogen interactions, virulence level, and early detection time points. Additionally, a comprehensive analysis of virulent Foc1_C2HIR and FocTR4_C1HIR isolates yielded informative genomic insights. Hence, these discoveries contribute to our comprehension of potential disease control targets in these plants, as well as enhancing plant diagnostics and breeding programs.

Onion (Allium cepa, L) is a very important vegetable crop in India. India is the second largest producer of onion in the world and the crop is grown on more than 1.22 million hectares. Fusarium Basal Rot (FBR) is an economically important diseasef onion that causes considerable losses in onion production up to 50% in field and 30-40% during post-harvest storage of bulbs (Gupta and Gupta 2013; Rajamohan et al. 2019). Onion plants showing chlorosis, twisting, wilting, necrosis, bulb discoloration, rot in the basal parts of bulb and roots typical to FBR were observed, in a field trial of 36 onion cultivars during October 2020 in Bangalore, Karnataka. FBR incidence varied from 30-100% in this field (Fig 1 a-d). Symptomatic bulbs were washed with water, basal plate and fleshy leaves cut into 0.5 to 1 cm-size, surface disinfected with 1.5% sodium hypochlorite (NaOCl) for 3 min, and rinsed with sterile distilled water. Twenty pieces were placed on potato dextrose agar (PDA) in Petri plates and incubated at 25°C for 7 days. Colonies from single-spore isolates on PDA showed abundant white aerial mycelium. Colonies showed light pink or purple coloration on the reverse side of the culture plate with brown center (Fig 1e-f). Macroconidia were 19.13 to 28.35 (mean= 24.2) × 4.29 to 6.06 (mean= 5.05) µm, hyaline, falcate, with slightly curved apexes, and three to five septa. Microconidia were cylindrical to ellipsoid, aseptate, hyaline 8.20 to 12 (mean=10.0) × 3.55 to 4.79 (mean= 4.29) µm (Fig 1g). Chlamydospores were round, intercalary, hyaline, single or in chains (Fig 1h). Two isolates (IBFF-09 & IBFF-10) were analyzed for internal transcribed spacer-ITS (White et al. 1990) and translation elongation factor 1-α (tef1) gene (O'Donnell et al. 1998) by polymerase chain reaction (PCR) and sequenced. ITS and partial tef1 gene sequences of isolates IBFF-09 and IBFF-10 were submitted to the NCBI database (GenBank accession # ON394614&ON026859; # ON409480, ON093166 respectively). Phylogenetic analysis of tef1 gene placed the isolates with F. falciforme (Fig 1i). A pathogenicity test was performed by dipping roots of 28 days old healthy onion seedlings of a susceptible genotype 16/7Y GR3 into a conidial suspension (1 × 104 conidia/ml) of isolate IBFF-10 for 15 min and then transplanting the plants into pots containing sterilized potting mix. Inoculated plants developed typical symptoms of FBR and were all dead by 20 days post inoculation (Fig 1j) while the non-inoculated controls remained healthy. Pathogen was re-isolated from infected plants and showed the same morphology, ITS and tef1 sequence similarity as the original isolate, thus fulfilling Koch's postulates. Basal rot of onion by F. falciforme is reported from Mexico (Tirado-Ramírez et al. 2021). Till date, only F .oxysporum, F. proliferatum and F. solani have been implicated in onion FBR in India (Lee et al. 2021; Rathore and Patil, 2019). F. falciforme however, i prevalent in India and is reported to infect other crops (Gangaraj et al. 2022; Gupta et al. 2019; Homa et al. 2018). There is a high probability that this pathogen is contributing significantly to basal rot disease but it has not been reported yet. To our knowledge, this is the first report of F. falciforme infecting onions in India. In order to develop FBR resistant onion cultivars it is critical to identify and study the response of onion genotypes to different Fusarium spp causing the disease.

SummaryThe FTF (Fusarium transcription factor) gene family comprises a single copy gene, FTF2, which is present in all the filamentous ascomycetes analysed, and several copies of a close relative, FTF1, which is exclusive to Fusarium oxysporum. An RNA‐mediated gene silencing system was developed to target mRNA produced by all the FTF genes, and tested in two formae speciales: F. oxysporum f. sp. phaseoli (whose host is common bean) and F. oxysporum f. sp. lycopersici (whose host is tomato). Quantification of the mRNA levels showed knockdown of FTF1 and FTF2 in randomly isolated transformants of both formae speciales. The attenuation of FTF expression resulted in a marked reduction in virulence, a reduced expression of several SIX (Secreted In Xylem) genes, the best studied family of effectors in F. oxysporum, and lower levels of SGE1 (Six Gene Expression 1) mRNA, the presumptive regulator of SIX expression. Moreover, the knockdown mutants showed a pattern of colonization of the host plant similar to that displayed by strains devoid of FTF1 copies (weakly virulent strains). Gene knockout of FTF2 also resulted in a reduction in virulence, but to a lesser extent. These results demonstrate the role of the FTF gene expansion, mostly the FTF1 paralogues, as a regulator of virulence in F. oxysporum and suggest that the control of effector expression is the mechanism involved.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is a major factor limiting banana production in Malaysia. To date, SSR, ISSR, AFLP and RAPD markers are widely used in PCR-based molecular characterization of Foc. Although these markers are effective in characterizing Foc, they all have their own limitations. An effector gene known as Secreted in Xylem (SIX) has been explored to broaden the molecular diagnostic toolbox for Foc. This study aims to investigate the diversity of Foc-TR4 in Peninsular Malaysia based on the phylogenetic analysis of SIX sequences. The virulence potential of twenty-seven Fusarium wilt of banana based on pathogenicity test and the presence of SIX1, SIX7 and SIX8 genes were determined. All of twenty-seven Foc ‘Tropical Race 4’ (TR4) (VCG 01213/160) isolates were obtained from Biological Control Laboratory, Department of Plant Protection, Universiti Putra Malaysia (UPM). The results showed that all Foc-TR4 isolates were pathogenic towards banana plantlets at different severity levels under greenhouse condition. Pathogenicity assays showed that the level of aggressiveness differs between isolates. The Foc-TR4 isolates were screened for the presence of three SIX genes (SIX1, SIX7 and SIX8) and the genetic differentiation of Foc-TR4 was evaluated by PCR analysis using three specific primers. Among these three SIX genes, SIX1 and SIX8 genes were detected in all twenty-seven Foc-TR4 isolates from Peninsular Malaysia. Phylogenetic analysis using SIX1 and SIX8 sequences showed that they were related to TR4 isolates (VCG 01213/16) from Australia and Indonesian with bootstrap values of 94% and 100% respectively. No variation was observed in the SIX1 and SIX8 sequences since all 27 isolates were clustered into the same clade. Virulence level also did not correlate with the existence or lack of these genes. Since Foc could hamper the local banana production in Malaysia, characterization of these isolates through SIX genes provides a great understanding of pathogenicity in Foc and could potentially improve the diagnostic and control of Fusarium wilt in the future.

In recent years, greenhouse-grown tomato (Solanum lycopersicum) plants showing vascular wilt and yellowing symptoms have been observed between 2015 and 2018 in North Carolina (NC) and considered as an emerging threat to profitability. In total, 38 putative isolates were collected from symptomatic tomatoes in 12 grower greenhouses and characterized to infer pathogenic and genomic diversity, and mating-type (MAT) idiomorphs distribution. Morphology and polymerase chain reaction (PCR) markers confirmed that all isolates were Fusarium oxysporum f. sp. lycopersici (FOL) and most of them were race 3. Virulence analysis on four different tomato cultivars revealed that virulence among isolates, resistance in tomato cultivars, and the interaction between the isolates and cultivars differed significantly (P < 0.001). Cultivar ‘Happy Root’ (I-1, I-2, and I-3 genes for resistance) was highly resistant to FOL isolates tested. We sequenced and examined for the presence of 15 pathogenicity genes from different classes (Fmk1, Fow1, Ftf1, Orx1, Pda1, PelA, PelD, Pep1, Pep2, eIF-3, Rho1, Scd1, Snf1, Ste12, and Sge1), and 14 Secreted In Xylem (SIX) genes to use as genetic markers to identify and differentiate pathogenic isolates of FOL. Sequence data analysis showed that five pathogenicity genes, Fmk1, PelA, Rho1, Sge1, and Ste12 were present in all isolates while Fow1, Ftf1, Orx1, Peda1, Pep1, eIF-3, Scd1, and Snf1 genes were dispersed among isolates. Two genes, Pep2 and PelD, were absent in all isolates. Of the 14 SIX genes assessed, SIX1, SIX3, SIX5, SIX6, SIX7, SIX8, SIX12, and SIX14 were identified in most isolates while the remaining SIX genes varied among isolates. All isolates harbored one of the two mating-type (MAT-1 or MAT-2) idiomorphs, but not both. The SIX4 gene was present only in race 1 isolates. Diversity assessments based on sequences of the effector SIX3- and the translation elongation factor 1-α encoding genes SIX3 and tef1-α, respectively were the most informative to differentiate pathogenic races of FOL and resulted in race 1, forming a monophyletic clade while race 3 comprised multiple clades. Furthermore, phylogeny-based on SIX3- and tef1-α gene sequences showed that the predominant race 3 from greenhouse production systems significantly overlapped with previously designated race 3 isolates from various regions of the globe.

Several Fusarium species cause disease in onion ( Allium cepa ), resulting in significant yield losses. In this study, isolates of Fusarium oxysporum and Fusarium proliferatum from diseased and symptomless mature onion bulbs were tested for pathogenicity on onion seedlings and mature bulbs. For F . oxysporum isolates, the outcome of the test on mature bulbs correlated well with the symptom status of the original bulb, whereas for F . proliferatum , such a correlation was not seen, suggesting a different mechanism of symptom development. Those F . oxysporum isolates that carried the Secreted In Xylem ( SIX ) genes, CRX genes and C5 gene were significantly more aggressive on both seedlings and mature bulbs. However, the SIX‐ negative F . oxysporum isolates also significantly reduced the emergence of onion seedlings, and thus a division into pathogenic and nonpathogenic F . oxysporum isolates was not obvious. On mature bulbs, those SIX ‐negative isolates that carried the gene CRX2 were significantly more virulent than those without CRX2 . The F . proliferatum isolates were all pathogenic to onion and carried one SIX2 gene homologue, SIX2‐1 . The majority of F . proliferatum isolates also carried another homologue, SIX2‐2 . Several isolates negative for SIX2‐2 had another potential virulence gene, CRX2 , suggesting that these two genes could encode overlapping functions. The SIX2‐2 sequence was identical in all those isolates harbouring it, while SIX2‐1 showed variation that agreed with the phylogeny based on the translation elongation factor 1‐α sequence. This suggests that the two SIX2 genes of F . proliferatum might have been acquired separately.

Over the past decade, there have been accumulating reports from researchers, farmers, and field extension personnel on the increasing incidence and spread of onion basal rot in India. Onion basal rot disease is mainly caused by Fusarium spp. This study aimed to validate the information on the active prevalence of F. falciforme and F. acutatum causing Fusarium basal rot (FBR) in Maharashtra. A survey was conducted, and the infected plants/bulbs were collected from fields of 38 locations comprising five districts of Maharashtra, namely, Nashik, Aurangabad, Solapur, Ahmednagar, and Pune, in 2023. This disease was prevalent in high-moisture and high-oil-temperature conditions and the symptoms were observed in most of the fields, with the FBR incidence ranging from 17 to 41%. The available data of basal rot incidence from 1998 to 2022 were analyzed, based on which the prevalence of FBR was 11–50%. Tissue from the infected samples of onion bulbs was used for the isolation. The identification was performed based on colony morphology and microscopic features and confirmed through molecular markers using ITS and Tef-1α gene primers. Of the ten Fusarium isolates collected from selected locations, six species were confirmed as F. acutatum and four as F. falciforme. The pathogenicity tests performed with onion seedlings and bulbs under moist conditions proved that both F. acutatum and F. falciforme independently could cause basal rot disease symptoms but with different degrees of virulence. Koch’s postulates were confirmed by reisolating the same pathogens from the infected plants. Thus, the active prevalence of FBR was confirmed in Maharashtra and also, to the best of our knowledge, this is the first report of F. falciforme and F. acutatum causing basal rot of onion independently in Maharashtra, India.

Putative effector genes detected in <i>Fusarium oxysporum</i> from natural ecosystems of Australia