Biological Control of Fusarium oxysporum, the Causal Agent of Fusarium Basal Rot in Onion by Bacillus spp.

Fusarium oxysporum is the main pathogen causing Fusarium basal rot in onion (Allium cepa L.), which incurs significant yield losses before and after harvest. Among management strategies, biological control is an environmentally safe and sustainable alternative to chemical control. In this study, we isolated and screened bacteria for antifungal activity against the basal rot pathogen F. oxysporum. Isolates 23-045, 23-046, 23-052, 23-055, and 23-056 significantly inhibited F. oxysporum mycelial growth and conidial germination. Isolates 23-045, 23-046, 23-052, and 23-056 suppressed the development of Fusarium basal rot in both onion seedlings and bulbs in pot and spray inoculation assays. Isolate 23-055 was effective in onion seedlings but exhibited weak inhibitory effect on onion bulbs. Based on analyses of the 16S rRNA and rpoB gene sequences together with morphological analysis, isolates 23-045, 23-046, 23-052, and 23-055 were identified as Bacillus thuringiensis, and isolate 23-056 as Bacillus toyonensis. All five bacterial isolates exhibited cellulolytic, proteolytic, and phosphate-solubilizing activity, which may contribute to their antagonistic activity against onion basal rot disease. Taken together B. thuringiensis 23-045, 23-046, 23-052, and 23-055 and B. toyonensis 23-056 have potential for the biological control of Fusarium basal rot in onion.

Identification of Fusarium Basal Rot Pathogens of Onion and Evaluation of Fungicides against the Pathogens.

Onion (Allium cepa L.) is an economically important vegetable crop worldwide. However, various fungal diseases, including Fusarium basal rot (FBR), neck rot, and white rot, reduce onion production or bulb storage life. FBR caused by Fusarium species is among the most destructive onion diseases. In this study, we identified Fusarium species associated with FBR in Jeolla and Gyeongsang Provinces in South Korea and evaluated fungicides against the pathogens. Our morphological and molecular analyses showed that FBR in onions is associated with Fusarium commune, Fusarium oxysporum, and Fusarium proliferatum. We selected seven fungicides (fludioxonil, hexaconazole, mandestrobin, penthiopyrad, prochloraz-manganese, pydiflumetofen, and tebuconazole) and evaluated their inhibitory effects on mycelial growth of the pathogens at three different concentrations (0.01, 0.1, and 1 mg/mL). We found that prochloraz-manganese was highly effective, inhibiting 100% of the mycelial growth of the pathogens at all concentrations, followed by tebuconazole. Fludioxonil showed < 50% inhibition at 1 mg/mL for the tested isolates.

First report of fusarium wilt disease caused by Fusarium equiseti on grafted watermelon in Korea.

In Korea, most of the grafted watermelons are a fusion of bottle gourd (Lagenaria siceraria) as a rootstock and watermelon (Citrullus lanatus) as a scionstock (Lee et al., 2010). Currently, we have collected several samples from grafted watermelon displaying symptoms of yellowing, withered and wilting leaves. When the symptomatic stem was excised, browning vascular tissues were observed due to the colonization of fungal pathogen. From the samples obtained, 25 fungal isolates were identified as species of Fusarium. Among 25 isolates, 18 were identified as Fusarium oxysporum, four as Fusarium solani, and three as Fusarium equiseti (F. equiseti) . Initial assessment showed that one of the F. equiseti isolates (NIHHS 16-126) was highly virulent to rootstock. Interestingly, this is the first time F. equiseti has been identified pathogenic to grafted watermelon. NIHHS 16-126 isolate was collected from watermelon cultivation field around Buyeo-gun (36.25951°N, 126.92044°E) county. Disease incident was estimated to infect approximately 10% of the watermelon plants cultivated in this area. NIHHS 16-126 isolate was examined to confirm its identity. On potato dextrose agar, colonies appeared yellowish-brown while the aerial mycelium was whitish to peach in color. Macroconidia were relatively long (20.21 - 51.13 × 2.30 - 4.5 µm, n=50), comprise of 3-6 septa, curvature shape and its conidiophores were with monophialides. However, microconidia formation was not observed. These morphological characteristics resemble F. equiseti characters as described by Hyun (2019). For molecular identification, an internal transcribed spacer of ribosomal DNA (ITS-rDNA), elongation factor-1α (EF-1α), and beta-tubulin (ß-tub) genes were sequenced using primer pairs of ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Glass and Donaldson 1995), and Bt2a/Bt2b (Carbone and Kohn 1999). BLASTN analysis revealed that ITS-rDNA (LC648248), EF-1α (LC648250), and ß-tub (LC648249) sequences were 99-100% identical to F. equiseti reference sequences (KF515650, KF747331, and KF747330) infected Avicennia marina in China (Lu 2014). Phylogenetic analysis of concatenated ITS-rDNA, EF-1α and ß-tub sequences showed that this isolate clustered in the same clade as F. equiseti, confirming its identity as F. equiseti. For the inoculation, roots of 12-days-old seedlings (watermelon and bottle gourd, n=10 each) were dipped in the conidia suspension (1x106 conidia/µL) for 30 min. Inoculated seedlings were planted in the soil before being transferred to the greenhouse (temperature; 30°C, daylight; 14 hours). Control plants were inoculated with sterile water. Results showed that after 21 days post-inoculation, all inoculated bottle gourd seedlings (n=10) wilted and eventually died. In contrast, none of the inoculated watermelons or control seedlings were affected. Re-isolation of three fungal isolates (infected root) showed that their morphology and gene markers sequence were identical to the original isolates thus fulfilled Koch's postulates. Bottle gourd is the most preferred rootstock for grafted watermelons among Korean farmers due to its ability to resist Fusarium spp. infection. Therefore, the identification of F. equiseti as a fungal that is pathogenic to rootstock is crucial information to manage fusarium wilt disease among grafted watermelon. To our knowledge, this is the first report confirming F. equiseti infection in grafted watermelon plants in Korea.

First report of Botryosphaeria dothidea as a causal agent to stem rot disease on plumcot trees in Korea.

Botryosphaeria dothidea (B. dothidea) is a fungal pathogen commonly associated with stem canker, dieback, and rot disease in a variety of woody plants worldwide (Dong and Guo, 2020). In Korea, B. dothidea was reported to cause a disease problem to serval crops such as apple and blueberry (Kim, 1995; Choi, 2011). In early 2020, a typical symptom resembling the stem rot disease was spotted to occur at a plumcot cultivation area around Wanju (35.827870, 127.030380) province, Korea. At the early stage of infection, a small blister appeared on the plumcot branch and stem. However, as the blister extended, a light brown canker was observed appeared on the infected area and in some cases a sticky sap oozed from the branch bark crack. If not managed or treated properly, all leaves beyond the infection site will turn brown, wilt, and the whole plumcot tree eventually dies. A survey in the affected area showed that approximately 5% of the plumcot trees were infected which cause up to 10% reduction in total production. To identify the causal agent, symptomatic tissues were excised and surface sterilized with 70% ethanol for 30 sec followed by 1% NaClO for 30 sec before rinsing with sterile water, thrice. The samples were then dried with a piece of filter paper and later air-dried before being placed on a potato dextrose agar (PDA). The PDA plates were then incubated at 25°C for 5 days with 12 hours light/dark cycles period. Among several fungal isolates obtained, four were selected for further analyses. Morphological identification revealed that the fungal conidia were hyaline, ovoid, fusiform (type that rarely form a septum) and unicellular with an average size of 18 - 20 µm × 4.5 -5.5 µm (n = 50). These morphological characters have a strong resemblance to B. dothidea that described by Slipper et al., (2004). For molecular identification, Internal transcribed spacer (ITS), beta-tubulin (ß-tubulin) and elongation factor 1 alpha (EF-1α) were amplified and sequenced using universal primer pairs ITS1/ITS4 (White et al., 1990), Bt2a/Bt2b (Glass and Donaldson, 1995) and EF1/EF2 (O'Donnell et al. 1998) respectively. Alignment analysis showed that ITS (LC602817), ß-tubulin (LC602820) and EF-1α (LC602821) sequences were 99-100% identical to the orthologous genes identified in B. dothidea infecting soybean in China [MW130133 (identity 537/536 bp), MW147482 (identity 394/394 bp) and MW147481 (identify 250/250 bp) respectively] (Chen et al. 2021). However, phylogenetic analysis of concatenated ITS, ß-tubulin and EF-1α genes sequence established the identity of these isolate as B. dothidea. Due to the 100% identical at the molecular level, isolate NIHHS 20-262 was selected as a representative for further analysis. For the pathogenicity test, fungal mycelium (via PDA plug) was used as a source of inoculum for both intact and detached plumcot stems trials. For the intact trial, mycelium was inoculated on the wounded spots of ten plumcot stems that grew at the NIHHS trial farm. Ten days post-inoculation (dpi), disease symptoms i.e. stem colour turn from greenish to dark brown was observed at the inoculated sites. For the detached trial, mycelium was inoculated on the wounded spots of ten detached plumcot stems. The inoculated stems were kept in a closed container to maintain 90% humidity before incubated at 25ºC in the dark. Interestingly, on the detached stems, disease symptoms (greenish colour turn to dark brown) were observed to appear seven days early compare to intact stems. A sterile PDA plug replacing fungal mycelium served as a negative control and the result shows no symptoms were observed on either intact or detached control stems. For consistency purposes, pathogenicity tests on intact stems were performed on three different plumcot trees, whereas three biological replicates for detached stems. Isolation and re-identification of two colonies from the infected sites (intact and detached stems) were attempted and the results obtained were identical to the original isolate, thus fulfilling Koch's postulates. Local farmers described this disease as a "certain death disease" in plumcot. Therefore, accurate identification of B. dothidea as the causal agent is critical for effective disease management to minimise qualitative and quantitative losses in the plumcot industry. Although has been reported to cause dieback disease in blueberry in Korea (Choi, 2011), to our knowledge, this is the first study to report B. dothidea causing stem rot diseases on the plumcot trees in Korea.

Transcriptome analyses of the ginseng root rot pathogens Cylindrocarpon destructans and Fusarium solani to identify radicicol resistance mechanisms.

BACKGROUND: The ascomycete fungi Cylindrocarpon destructans (Cd) and Fusarium solani (Fs) cause ginseng root rot and significantly reduce the quality and yield of ginseng. Cd produces the secondary metabolite radicicol, which targets the molecular chaperone Hsp90. Fs is resistant to radicicol, whereas other fungal genera associated with ginseng disease are sensitive to it. Radicicol resistance mechanisms have not yet been elucidated. METHODS: Transcriptome analyses of Fs and Cd mycelia treated with or without radicicol were conducted using RNA-seq. All of the differentially expressed genes (DEGs) were functionally annotated using the Fusarium graminearum transcript database. In addition, deletions of two transporter genes identified by RNA-seq were created to confirm their contributions to radicicol resistance. RESULTS: Treatment with radicicol resulted in upregulation of chitin synthase and cell wall integrity genes in Fs and upregulation of nicotinamide adenine dinucleotide dehydrogenase and sugar transporter genes in Cd. Genes encoding an ATP-binding cassette transporter, an aflatoxin efflux pump, ammonium permease 1 (mep1), and nitrilase were differentially expressed in both Fs and Cd. Among these four genes, only the ABC transporter was upregulated in both Fs and Cd. The aflatoxin efflux pump and mep1 were upregulated in Cd, but downregulated in Fs, whereas nitrilase was downregulated in both Fs and Cd. CONCLUSION: The transcriptome analyses suggested radicicol resistance pathways, and deletions of the transporter genes indicated that they contribute to radicicol resistance.

Improved Vision-Based Detection of Strawberry Diseases Using a Deep Neural Network.

Detecting plant diseases in the earliest stages, when remedial intervention is most effective, is critical if damage crop quality and farm productivity is to be contained. In this paper, we propose an improved vision-based method of detecting strawberry diseases using a deep neural network (DNN) capable of being incorporated into an automated robot system. In the proposed approach, a backbone feature extractor named PlantNet, pre-trained on the PlantCLEF plant dataset from the LifeCLEF 2017 challenge, is installed in a two-stage cascade disease detection model. PlantNet captures plant domain knowledge so well that it outperforms a pre-trained backbone using an ImageNet-type public dataset by at least 3.2% in mean Average Precision (mAP). The cascade detector also improves accuracy by up to 5.25% mAP. The results indicate that PlantNet is one way to overcome the lack-of-annotated-data problem by applying plant domain knowledge, and that the human-like cascade detection strategy effectively improves the accuracy of automated disease detection methods when applied to strawberry plants.

Roles of the glyoxylate and methylcitrate cycles in sexual development and virulence in the cereal pathogen Gibberella zeae.

The glyoxylate and methylcitrate cycles are involved in the metabolism of two- or three-carbon compounds in fungi. To elucidate the role(s) of these pathways in Gibberella zeae, which causes head blight in cereal crops, we focused on the functions of G. zeae orthologs (GzICL1 and GzMCL1) of the genes that encode isocitrate lyase (ICL) and methylisocitrate lyase (MCL), respectively, key enzymes in each cycle. The deletion of GzICL1 (DeltaGzICL1) caused defects in growth on acetate and in perithecium (sexual fruiting body) formation but not in virulence on barley and wheat, indicating that GzICL1 acts as the ICL of the glyoxylate cycle and is essential for self-fertility in G. zeae. In contrast, the DeltaGzMCL1 strains failed to grow on propionate but exhibited no major changes in other traits, suggesting that GzMCL1 is required for the methylcitrate cycle in G. zeae. Interestingly, double deletion of both GzICL1 and GzMCL1 caused significantly reduced virulence on host plants, indicating that both GzICL1 and GzMCL1 have redundant functions for plant infection in G. zeae. Thus, both GzICL1 and GzMCL1 may play important roles in determining major mycological and pathological traits of G. zeae by participating in different metabolic pathways for the use of fatty acids.

A novel F-box protein involved in sexual development and pathogenesis in Gibberella zeae.

Gibberella zeae is an ascomyceteous fungus that causes serious diseases in cereal crops. Severe epidemics require strains that are virulent and that can reproduce sexually. We characterized an insertional mutant (designated ZH436) with a pleiotropic defect in both traits, and identified a novel F-box protein gene encoding FBP1 (F-box protein 1) that is similar to fungal F-box proteins including Saccharomyces cerevisiae Grr1, a well-characterized component of the Skp1-Cullin-F-box protein (SCF(Grr1)) E3 ligase complex required for protein degradation. FBP1 also can bind both S. cerevisiae Skp1 protein, the other component of the SCF(Grr1) complex, and its G. zeae sequence homologue SKP1. Two putative protein interacting domains in FBP1 are essential for in vivo function. FBP1 and ScGRR1 are not so interchangeable between S. cerevisiae and G. zeae, but FBP1 can partially complement several defects of a yeast grr1 deletion mutant. Functional analyses confirmed that FBP1 is required for several phenotypes including both sexual development and virulence in G. zeae; the phenotype of DeltaFBP1 strains is different from those of null mutants for F-box proteins in other filamentous fungi as well as from S. cerevisiae grr1Delta strains. Thus, FBP1 is a versatile F-box protein that presumably participates in the formation of the SCF(FBP1) complex that probably controls the ubiquitin-mediated degradation of proteins involved in sexual reproduction and virulence important for disease development by G. zeae.

Functional analysis of the homoserine O-acetyltransferase gene and its identification as a selectable marker in Gibberella zeae.

We used restriction enzyme-mediated integration (REMI) to identify a methionine auxotrophic mutant of Gibberella zeae, an important cereal pathogen. In addition to its methionine requirement, the G. zeae REMI mutant designated Z43R3912 showed pleiotropic phenotypes, including reduced virulence on host plants and lack of sexual development. Outcrossing of Z43R3912 with a mat1-1 deletion strain confirmed that the mutation of Z43R3912 was tagged with the hygromycin B resistance marker. The vector insertion site in Z43R3912 was identified within the ORF designated GzmetE, encoding a putative homoserine O-acetyltrasferase (HOA). Gene disruption analyses confirmed that GzmetE was responsible for the pleiotropic phenotypes of Z43R3912. Genetic complementation of the G. zeae methionine auxotroph with an intact copy of the Aspergillus nidulans metE and GzmetE genes suggests that the HOA gene can be used as a selectable marker for transformation of G. zeae.

Tri13 and Tri7 determine deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae.

Gibberella zeae, a major cause of cereal scab, can be divided into two chemotypes based on production of the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced a Tri13 homolog from each chemotype. The Tri13 from a NIV chemotype strain (88-1) is located in the trichothecene gene cluster and carries an open reading frame similar to that of Fusarium sporotrichioides, whereas the Tri13 from a DON chemotype strain (H-11) carries several mutations. To confirm the roles of the Tri13 and Tri7 genes in trichothecene production by G. zeae, we genetically altered toxin production in 88-1 and H-11. In transgenic strains, the targeted deletion of Tri13 from the genome of 88-1 caused production of DON rather than NIV. Heterologous expression of the 88-1 Tri13 gene alone or in combination with the 88-1 Tri7 gene conferred on H-11 the ability to synthesize NIV; in the latter case, 4-acetylnivalenol (4-ANIV) also was produced. These results suggest that Tri13 and Tri7 are required for oxygenation and acetylation of the oxygen at C-4 during synthesis of NIV and 4-ANIV in G. zeae. These functional analyses of the Tri13 and Tri7 genes provide the first clear evidence for the genetic basis of the DON and NIV chemotypes in G. zeae.