Oxysporum Isolates Research Articles

Identification of Fusarium Wilt Disease Causal Agents in Pistachio Fields and Determination of Efficacy of Some Fungicides Controlling of The Disease

Pistachio (Pistacia vera) propagation is commonly carried out by seedlings and new orchards are mostly set by in situ grafting of rootstocks. Pathogenic and molecular characterization of wilt and desiccation agents observed in nurseries and young orchards were carried out and the causal species were identified as Fusarium oxysporum and F. solani. In vitro inhibition of mycelial development and conidia germination were carried out with seven fungicides selected against the disease. The plant protection product containing Prothioconazole + Spiroxamine was found to be 100% effective in mycelial growth trials in F. oxysporum isolates from 20 mgL-1 and in F. solani from 100 mL-1 at all increasing doses. . The fungicide containing phosphoric acid was found to be 18.3%-33.3% effective on both species at 2.5 times the recommended dose. On conidial germination, Prothioconazole + Spiroxamine and Captan provided 100% inhibition even at the lowest doses in all isolates tested. The EC50 values of Prothioconazole + Spiroxamine, which was determined as the most effective chemical in mycelial development and conidial germination, were 3.67 ml and 3.72 ml in F. oxysporum isolates and 56.83 ml in F. solani, respectively.

A systematic approach for identifying unique genomic sequences for Fusarium oxysporum f. sp. lactucae race 1 and development of molecular diagnostic tools

Fusarium oxysporum f. sp. lactucae (FOLac) is a soil- and seedborne fungal pathogen that causes Fusarium wilt of lettuce, an important disease threatening global lettuce production. Based on pathogenicity on differential lettuce cultivars, four races (1-4) have been identified, with race 1 the only race detected in the United States, and the closely related, emerging race 4 known only in Europe. The development of race-specific diagnostic tools is hindered by insufficient genomic data to distinguish between the two races and FOLac from other F. oxysporum formae speciales and nonpathogenic isolates. Here, we describe a systematic approach for developing diagnostic markers for FOLac race 1 that utilized a comprehensive sequence database of F. oxysporum to identify 15 unique genomic sequences. Marker specificity was validated through an exhaustive screening process against genomic data from 797 F. oxysporum isolates representing 64 formae speciales and various plants and non-plant substrates. One of the unique sequences was used to develop a TaqMan quantitative polymerase chain reaction assay and a recombinase polymerase amplification assay, both exhibiting 100% sensitivity and specificity when tested against purified DNA from 171 F. oxysporum isolates and 69 lettuce samples. The relationship between qPCR Ct values and colony forming units (CFU)/g values was also determined. This study not only introduces a new marker for FOLac race 1 diagnostics and soil quantitation, but also underscores the value of an extensive genomic database and screening software pipeline for developing molecular diagnostics for F. oxysporum formae speciales and other fungal taxa.

Assessing the Pathogenicity of Berkeleyomyces rouxiae and Fusarium oxysporum f. sp. vasinfectum on Cotton (Gossypium hirsutum) Using a Rapid and Robust Seedling Screening Method.

Cotton (Gossypium spp.) is the most important fibre crop worldwide. Black root rot and Fusarium wilt are two major diseases of cotton caused by soil-borne Berkeleyomyces rouxiae and Fusarium oxysporum f. sp. vasinfectum (Fov), respectively. Phenotyping plant symptoms caused by soil-borne pathogens has always been a challenge. To increase the uniformity of infection, we adapted a seedling screening method that directly uses liquid cultures to inoculate the plant roots and the soil. Four isolates, each of B. rouxiae and Fov, were collected from cotton fields in Australia and were characterised for virulence on cotton under controlled plant growth conditions. While the identities of all four B. rouxiae isolates were confirmed by multilocus sequencing, only two of them were found to be pathogenic on cotton, suggesting variability in the ability of isolates of this species to cause disease. The four Fov isolates were phylogenetically clustered together with the other Australian Fov isolates and displayed both external and internal symptoms characteristic of Fusarium wilt on cotton plants. Furthermore, the isolates appeared to induce varied levels of plant disease severity indicating differences in their virulence on cotton. To contrast the virulence of the Fov isolates, four putatively non-pathogenic Fusarium oxysporum (Fo) isolates collected from cotton seedlings exhibiting atypical wilt symptoms were assessed for their ability to colonise cotton host. Despite the absence of Secreted in Xylem genes (SIX6, SIX11, SIX13 and SIX14) characteristic of Fov, all four Fo isolates retained the ability to colonise cotton and induce wilt symptoms. This suggests that slightly virulent strains of Fo may contribute to the overall occurrence of Fusarium wilt in cotton fields. Findings from this study will allow better distinction to be made between plant pathogens and endophytes and allow fungal effectors underpinning pathogenicity to be explored.

A Novel Secreted Protein of Fusarium oxysporum Promotes Infection by Inhibiting PR-5 Protein in Plant.

Fusarium oxysporum, an important soilborne fungal pathogen that causes serious Fusarium wilt disease, secretes diverse effectors during the infection. In this study, we identified a novel secreted cysteine-rich protein, FolSCP1, which contains unknown protein functional domain. Here, we characterized FolSCP1 as a secreted virulence factor that promotes the pathogen infection of host plants by inhibiting diverse plant defence responses. FolSCP1 interacted with the pathogenesis-related 5 (PR-5) protein SlPR5, a positive regulator of tomato plant immunity against multiple tomato pathogens, and effectively attenuated the antifungal activity of the tomato PR-5 protein. FoSCP1, a homologue of FolSCP1, was secreted by a F. oxysporum isolate from infected tobacco and targeted the tobacco PR-5 protein NtPR5 to suppress plant defence for further infection. In summary, our study revealed a fungal virulence strategy in which F. oxysporum secrete effectors that interfere with plant immunity by binding to the PR-5 protein of the host plant and inhibiting its biological activity, thereby promoting fungal infection.

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 in banana plantations in Türkiye

important disease for banana production. Presence and prevalence of FocTR4 in banana plantations on the Mediterranean coast of Türkiye were assessed during 2018 to 2020 in a total of 117 banana plantations in open fields and protected plastic greenhouses. Rhizome, pseudostem, and root samples were taken from plants showing typical symptoms associated to the disease and from suspected affected plants. Fungi were isolated from the plant internal tissues, and Fusarium oxysporum-like colonies were sub-cultured for further analyses. Phylogenetic analyses of 36 isolates showed that they belonged to four different Fusarium species: F. musae, F. oxysporum, F. sacchari, and F. solani. Eight representative F. oxysporum isolates were identified as FocTR4 by specific PCR and qPCR tests. Pathogenicity tests were carried out on tissue-cultured ‘Cavendish’ type banana seedlings (‘Grand Naine’) for 36 Fusarium isolates, and their virulence was assessed based on the internal necrosis observed in the rhizomes. Approx. 40 to 65 d after inoculations, FocTR4, F. oxysporum, and F. sacchari isolates caused severe to mild necroses in the seedling rhizomes. This is the first report of F. sacchari associated with root and collar rot of bananas in Türkiye. This study showed that Fusarium wilt caused by FocTR4 is present, but at low incidence (6.8%) in Turkish banana plantations.

Molecular characterization of Secreted in Xylem 1 (Six1) gene of Fusarium oxysporum causing wilt of potato (Solanum tuberosum)

AbstractDeciphering effector genes of plant‐pathogenic fungi is key to understanding their role in pathogenicity because acquisition of such genes through horizontal transfer may result in emergence of new pathogenic races. A full‐length (837 bp) Six1 gene has been cloned and sequenced from the genome of Fusarium oxysporum FPo isolated from roots of wilted potatoes in West Bengal, India. The isolate was identified based on conidial morphology and sequencing of the 28S rDNA and elongation factor 1α gene. Koch's postulates established that the isolate could incite wilting in potato plants. The Six Gene Expression 1 gene (Sge1) was amplified from the isolate and compared with that of other wilt‐causing F. oxysporum isolates. The Six1 gene had no intron and it encoded 278 amino acids, with a calculated molecular weight of 30.6 kDa, estimated isoelectric point 5.66 and theoretical extinction coefficient of 57,410 M−1 cm−1. Multiple sequence alignment of SIX1 amino acid sequences showed that the length of SIX1 varied among F. oxysporum formae speciales and different isolates of the same forma specialis. The predicted secondary structure of FPoSIX1 contained four α‐helices and 14 β‐sheets. Its predicted tertiary structure showed 92% query coverage and 67.37% identity with the template of the SIX1 protein of F. oxysporum f. sp. lycopersici PDB code: 7t69. The QMEAN score of this model was −2.53 and the model was validated using the Ramachandran Phi/Psi plot. Structure analysis of the effector protein will be helpful to identify its specific function and recognition by the host resistance protein.

Incidence of fungal contamination in fresh ginseng samples and mycotoxigenic potential of representative fungal isolates.

Fresh ginseng is typically accompanied by soil after harvest, leading to contamination with harmful fungi during storage and distribution. In this study, we investigated the incidence of fungal contamination in fresh ginseng (5-6 years old) purchased from 22 different stores in Geumsan, Korea. The incidence of fungal contamination in the samples was 67.4-111.5%. Fusarium solani was the most abundant species in the head (38.5%) and fine root (19.3%) parts of the ginseng samples, whereas F. oxysporum was the most abundant in the main root (22.0%) part. We isolated Aspergillus, Fusarium and Penicillium spp. (total number of isolates: 395) from the ginseng samples, and 138 isolates were identified using phylogenetic analysis. Polymerase chain reaction-based screening of 65 mycotoxin-producing species revealed that two P. expansum isolates were positive for citrinin and/or patulin, and five F. oxysporum isolates were positive for fumonisin biosynthesis gene. One P. expansum isolate produced 738.0 mg kg-1 patulin, and the other produced 10.4 mg kg-1 citrinin and 12.0 mg kg-1 patulin on potato dextrose agar (PDA) medium. Among the 47 representative F. oxysporum isolates, 43 (91.5%) produced beauvericin (0.1-15.4 mg kg-1) and four of them (8.5%) produced enniatin B and enniatin B1 (0.1-1.8 mg kg-1) as well. However, none of these toxins was detected in fresh ginseng samples. Fusarium solani and F. oxysporum were the most abundant species in fresh ginseng samples. Most F. oxysporum (43) and P. expansum (2) strains isolated from fresh ginseng produced beauvericin and enniatins (B and B1), and patulin or citrinin, respectively, on PDA medium. This is the first report of the mycotoxigenic potential of P. expansum and F. oxysporum strains isolated from fresh ginseng. © 2024 Society of Chemical Industry.

Secreted in Xylem (SIX) Gene SIX9 Is Highly Conserved in Fusarium oxysporum f. sp. vasinfectum Race 4 Isolates from Cotton in the United States

Many Fusarium oxysporum formae speciales produce small, infection-dependent effector proteins called “secreted in xylem” (SIX) proteins. These proteins are secreted into the xylem of a plant during the infection process and are thought to promote virulence. In this study, a collection of Fusarium oxysporum isolates composed primarily of different races of Fusarium oxysporum f. sp. vasinfectum (FOV) was screened for the presence of 14 SIX effector genes ( SIX1 to SIX14). Our results showed that some of the most virulent FOV races, FOV4 and FOV7, share a common SIX effector: SIX9. This effector is largely absent in other FOV races in North America, making SIX9 a potential target for rapid detection of these highly virulent FOV strains and enabling race-specific FOV quantification in infected host plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Determination of fungal root and stem rot agents of melons grown in Kumluca/Antalya

Kumluca has an important place in terms of greenhouse vegetable cultivation. Melon is among one of the main vegetables grown in the district on about 3200 decare of land. Various diseases cause decrease in the yield and quality of melons grown undercover in Türkiye. Among them, Fusarium wilt and gummy stem blight diseases especially have caused significant losses in recent years. In this research, the incidence and severity of the root and stem rot disease in Kumluca were determined by surveys made in 72 melon greenhouses in this area. Plant and soil samples were taken to the laboratory and isolations were made. As a result, Fusarium oxysporum and Didymella bryoniae were the most frequently isolated pathogens from the plant samples, followed by other Fusarium species. Fungi with the highest isolation frequency from the soil samples were Fusarium spp., Rhizoctonia solani and Macrophomina phaseolina. In the pathogenicity test, F. oxysporum, F. solani, F. verticillioides, M. phaseolina and R. solani isolates caused severe symptoms on melon seedlings. Virulence of the F. oxysporum isolates on different cucurbit species was also investigated and it was determined that they caused severe wilting on melon and watermelon seedlings, while symptoms on squash and cucumber were moderate or slight. Additionally, reactions of five melon cultivars (Yusufbey, Çıtırex, Niovi, Ferdevs and Memory) commonly grown in the region against D. bryoniae were investigated using randomly selected four pathogen isolates. All the cultivars were susceptible to the disease.

Distribution of banana Fusarium wilt in Banyumas, Indonesia, and characterization of F. oxysporum isolates from infected bananas and taro growing on the same farm

Distribution of banana Fusarium wilt in Banyumas, Indonesia, and characterization of F. oxysporum isolates from infected bananas and taro growing on the same farm

Evaluation of Copper Nanoparticle Synthesis by Fusarium oxysporum Antibacterial Activity Against Staphylococcus aureus

The creation of effective, environmentally friendly antimicrobial alternatives is becoming more and more important in light of the development of antibiotic resistance. The objective of the present study was to illustrate the biosynthetic process of copper (II) carbonate nanoparticles, CuCo3 NPs, by using Fusarium oxysporum culture filtrate as an antibacterial activity against multidrug-resistant (MDR) clinical bacterial isolates, Staphylococcus aureus. Fusarium oxysporum isolate was diagnosed by PCR. One hundred and sixty specimens of pathogenic bacteria were collected from different sources (wounds, urine, sputum) then the bacterial isolates were diagnosed as Staphylococcus aureus by conventional morphological examination, biochemical tests, and the Vitek-2 system. Fusarium oxysporum culture filtrate was prepared by a culture of the fungus on Czapek Dox broth media modified by adding cornmeal and incubated for 14 days with shaking at 27±2°C and filtered in the last stages using a Millipore. The biosynthesis of CuCo3 NPs was done by adding 1g of CuCo3 Cu (OH)2 to 10 ml of Fusarium oxysporum culture filtrate. The NPs were diagnosed using modern methods, including UV-VIS, FT-IR, AFM, FE-SEM, and EDX techniques. Multidrug-resistant Staphylococcus aureus was tested on the produced Cu NPs. The results indicated that the prepared CuCo3 NPs inhibited pathogenic bacterial isolates of Staphylococcus aureus at concentration of 500, 250, 125, and 62.5 mg/ml.

First report of root and crown rot caused by Fusarium oxysporum Schltdl. on Prunus cerasifera L. in Spain.

In 2021, Spain was the largest producer of cherries in Europe with a production of 125810 tons (FAOSTAT, 2021). In May 2022, in several production fields in Huelva (Spain), wilting was noted in 4-year-old cherry trees cv. Crystal Champaign grafted on rootstock cv. Adara (Prunus cerasifera L.). Gumming and wilting affected approx. 1% of the production area, leading to the eventual collapse and death of most affected trees within 2-3 years. Discoloration of the vascular system of the crown and roots was also noted. Symptomatic crown and root pieces (0.5 cm) were subjected to surface sterilization: immersed in 1% NaClO for 2 min, rinsed in sterile distilled water, and air-dried in a laminar flow cabinet. Then, plant tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated in a lab bench at room temperature for a week. Cottony and pink colonies were observed growing from the tissues. Two single strains (F175 and F176) were obtained from each tree by excising single spores (Gordon and Okamoto 1991). Isolates produced sparse aerial mycelia with white to pinkish-orange pigmentation on Spezieller Nährstoffarmer Agar (SNA). Both isolates produced microconidia in false heads on short monophialides. Microconidia were hyaline and measured in the range of 5.0-17.5 × 2.5-3.8 µm for both isolates (n = 50). Macroconidia were less abundant, falciform, and hyaline. Morphological characteristics were consistent with identification as Fusarium spp. (Leslie and Summerell 2006). A portion of the translation elongation factor-1 alpha (EF-1α) gene was sequenced using EF1/2 primers (O'Donnell et al. 1998) (GenBank Accession Nos. OR733348 and OR733349). Based on a comparison of 619 base pairs (bp), both isolates exhibited different sequences, with a 99.5% similarity (616/619 bp). A comparison with previously described isolates revealed a 100% match with published F. oxysporum sequences in the GenBank database (KT323846 and MZ404079, respectively). Isolates were used to conduct pathogenicity tests on 1-year-old plants cv. Adara growing in 512 cm3 pots. Using a scalpel, a 6-7 mm-length wound (2-3 mm deep) was made 5 cm above the soil line in all plants. For each isolate, 5 plants were inoculated by placing a 5 mm plug containing 10-day-old mycelia grown in AMAP medium (Borrero et al. 2009) at the incision point. Non-colonized AMAP plugs were used to inoculate 5 control plants. The inoculated sites were sealed with parafilm. Plants were randomly placed in a growth chamber with a temperature of 28/24ºC and a 12-hour photoperiod. A reddish-brown vascular stem discoloration was noticed in all the inoculated plants 73 days after inoculation. On average, the length of the necrotic area was 12.73 cm for F175, 20.12 cm for F176, and 4.59 cm for the control plants. Fusarium oxysporum was successfully re-isolated from all the inoculated plants. Recovered isolates were confirmed to be the same as the inoculated ones by sequencing the EF-1α gene. A one-way ANOVA indicates that plants cv. Adara were susceptible to both F. oxysporum isolates (P < 0.05). This is particularly noteworthy as cherries are predominantly planted on rootstocks and cv. Adara is a widely used rootstock in Spain. While F. oxysporum has been reported as the cause of root and crown rot in sweet cherry (P. avium L.) in British Columbia (Úrbez-Torres et al. 2016), this is the first report of F. oxysporum causing root and crown rot in cherry rootstocks (P. cerasifera L.) in Spain.

Survey, Pathogenicity assay, and Molecular Identification of the Pathogenic Fungi Associated with Pistachio in Anbar Province, Iraq

This study was carried out in 6 different locations in the Anbar Province, between January and February 2021. The locations with higher pistachio cultivation, such as Kabisa, Al-Haditha, Al-Baghdadi, Anah, Al-Qaim, and Rawa were selected to study the prevalence of fungal diseases affecting pistachio crops. Twenty-six distinct fungal isolates were identified, and each isolate was examined for the pathogenicity. Although the level of pathogenicity of each isolate varied from high to moderate, they all caused lesions, damaged vascular tissue, signs of root rot and seed rot in pistachio seedlings. The fungal strains were identified depending on ITS marker. Twelve of the isolates belonged to Fusarium solani, 10 of F. falciforme isolates, 2 of F. oxysporum isolates, while 1 isolate of F. vanettenii and Xylaria sp. The most frequently-occurring species were F. falciforme and F. solani in the Anbar Province. Out of all the studied isolates, Al-Qaim and Al-Baghdadi were the highest pathogenic isolates. The pathogenic effects of F. vanettenii and Xylaria sp. on pistachio plants as well as the existence of F. solani and F. falciforme in Iraq were all recorded in this study for the first time.

Pathogenicity of Fusarium oxysporum f. sp. lycopersici (Sacc.) Isolates in Causing Tomato Wilt Disease on Two Tomato (Solanum lycopersicum L) Varieties

Pathogenicity test of Fusarium oxysporum isolates in causing tomato wilt disease (TWD) was carried out on two tomato varieties (UC 82B and Rio-grande) in a screen house located at the Teaching and Research Farm of Federal University of Agriculture, Makurdi during 2015 cropping season. The experiment was a 2 x 11 factorial laid out in Completely Randomized Design (CRD) and replicated three times. F. oxysporum isolates tested were coded as: FoAs1, FoAs2, FoAg, FoNb, FoSb, FoAm, FoAk, FoOr, FoAd and FoUAM together with an uninoculated control. All the isolates of F. oxysporum tested were pathogenic, causing wilt on the plants from 3 weeks after inoculation (WAI), with severity of wilt been significantly higher (P≤0.05) in FoUAM. Isolates of F. oxysporum showed significant difference (P≤0.05) both in incidence and severity compared with the control. Effect of F. oxysporum isolates on some agronomic characteristics such as plant height, number of fruits, fruit weight and number of branches on the two varieties of tomato at 12 weeks after sowing (WAS) was significantly different (P≤0.05) with the control experiment. Highest severity score of 5.00 was calculated in FoUAM while the least of 4.33 was in FoSb compare with uninoculated value of 2.50. It is therefore, concluded that the two tomato varieties are pathogenic to F. oxysporum isolates and illicit disease in tomato hence reduced the yield of the crop.

Characterization of field isolates of Fusarium spp. from eggplant in India for species complexity and virulence

Eggplant wilt, despite emerging as a severe disease in India, the etiology must be better studied for its species' complexity and variability. The identity of fungal isolates associated with eggplants of India was established morphologically followed by sequencing and phylogenetic analysis. Three species, Fusarium falciforme, Fusarium incarnatum and Fusarium proliferatum, were observed for the first time in India. The isolates were tested for pathogenicity. Though all of them were pathogenic, the isolates displayed varying degrees of virulence. In further studies, the genetic relatedness of the isolates for virulence was assessed with candidate avirulent (SIX effectors), virulent (Fow1 and Fow2) and SSR markers. The SIX effector genes could not delineate the virulent isolates and were expressed in some non-F. oxysporum isolates for the first time. Likewise, the virulent genes, Fow1 for expression across the isolates and Fow2 for random expression across the isolates, were unsuitable markers for identifying the virulent groups. Hence, the F. oxysporum and F. solani isolates were genotyped with SSR markers. Though the clustering did not correlate with their virulence levels, the dendrogram grouping revealed variability among the F. oxysporum and F. solani isolates. This study concludes that although multiple species of Fusarium are associated with eggplant wilt in India, only F. oxysporum and F. solani are widespread in the surveyed areas. Though the three markers could not delineate the race specificity of the isolates, only the SSR makers could identify the genetic variability and hence, would help screen eggplant germplasm for fusarium wilt resistance.

Specific members of the TOPLESS family are susceptibility genes for Fusarium wilt in tomato and Arabidopsis.

Vascular wilt diseases caused by Fusarium oxysporum are a major threat to many agriculturally important crops. Genetic resistance is rare and inevitably overcome by the emergence of new races. To identify potentially durable and non-race-specific genetic resistance against Fusarium wilt diseases, we set out to identify effector targets in tomato that mediate susceptibility to the fungus. For this purpose, we used the SIX8 effector protein, an important and conserved virulence factor present in many pathogenic F. oxysporum isolates. Using protein pull-downs and yeast two-hybrid assays, SIX8 was found to interact specifically with two members of the tomato TOPLESS family: TPL1 and TPL2. Loss-of-function mutations in TPL1 strongly reduced disease susceptibility to Fusarium wilt and a tpl1;tpl2 double mutant exerted an even higher level of resistance. Similarly, Arabidopsis tpl;tpr1 mutants became significantly less diseased upon F. oxysporum inoculation as compared to wildtype plants. We conclude that TPLs encode susceptibility genes whose mutation can confer resistance to F. oxysporum.

Phytochemical analysis and antifungal activity of Azadirachta indica and Balanites aegyptiaca seed extracts against Fusarium oxysporum isolate on tomatoes

Fusarium oxysporum is the causative agent of tomato wilt and is an important disease of tomato crops worldwide. Management through chemical fungicides causes serious environmental damage and human health. This study assesses the antifungal potential of biochemical compounds of Azadirachta indica and Balanites aegyptiaca seeds extracts identified by GC-MS analytical method against F. oxysporum. Acetone, methanol and aqueous extracts of A. indica and B. aegyptiaca at 15, 30 and 60 μL/mL concentrations and synthetic fungicide (3.33 g/L) were tested in vitro in triplicate. Mycelial growth and Minimum inhibitory concentrations (MIC50, MIC90) were evaluated 8 days after inoculation (DAI). As results, 55 and 34 phytochemical compounds were identified in seed extracts of A. indica and B. aegyptiaca respectively. Methanol extract of A. indica and B. aegyptiaca at 60 μL/mL concentration showed a total inhibition (100%) of F. oxysporum mycelial growth. Low MIC50 values were obtained with aqueous extract of A. indica (8.77 μL/mL) and methanol extract of B. aegyptiaca (17.91 μL/mL). The tentative action mode showed that the methanol extract of both plants induced wall lysis of F. oxysporum. These results suggest that A. indica and B. aegyptiaca seed extracts have potent antifungal activity and could be used as an alternative to control F. oxysporum. Keywords: Neem, desert date, bioactive compounds, mycelial growth, antifungal activity, Fusarium oxysporum

First Report of Fusarium oxysporum Causing Root Rot on Pleione bulbocodioides in China.

The members of Pleione (Orchidaceae) are popular worldwide due to their beautiful flowers and medicinal values. In October 2021, we observed the typical symptoms of yellow or brown leaves, rotted root, and plant death on P. bulbocodioides (Sup. S1a). Nearly 30% of the plants showed disease symptom in the farm in Zhaotong city, Yunnan Province, China. Three fresh root samples with typical symptoms were collected from the plants of P. bulbocodioides in the field. The root sections (3mm × 3mm) from the border of the symptomatic tissue were cut and sterilized with 75% ethanol for 30 s, followed by 3% sodium hypochlorite (NaClO) for 2 min, and then rinsed three times with sterile water. The sterilized root tissues were inoculated on potato dextrose agar (PDA) at 28℃ in the incubator for 3 days. The colonies were obtained and sub-cultured from the hyphal tip to the new PDA to further purify. The colonies grew up on PDA at 28℃ for 1 week, the hyphal color from white turned to purple, the center of colony became brick red. The colonies produced abundant microconidia, macroconidia and chlamydospores, but no sporodochia was observed (Sup. S2). The microconidia were oval and irregularly oval, zero to one septate, and measured 2.0 × 5.2 to 4.1 × 12.2 µm (n = 20). The macroconidia were falcate, slender, with a distinct curve to the latter half of the apical cell, three to five septate, and measured 4.0 × 15.2 to 5.1 × 39.3 µm (n = 20). Morphological characterization showed that the three isolates were similar, and appeared to be Fusarium oxysporum (Leslie and Summerell, 2006). For molecular identification, total genomic DNA of two representative isolates DSL-Q and DSL-Y were extracted with CTAB method, and the PCR amplification were performed. The sequence of partial elongation factor (TEF1-α) gene was amplified using the primer pair EF-1/EF-2 (O'Donnell et al. 1998). The sequence of β-tublin gene (TUB2) was amplified using the primer pair T1/T22 (O'Donnell and Cigelnik, 1997). The sequences from the two isolates were obtained and sequenced. Clustal2.1 searches indicated that the sequences of the three loci of the two isolates revealed 97.8% to 100%similarity to F. oxysporum strains and were deposited in GenBank (accession nos. OP150481 and OP150485 for TEF1-α; OP150483 and OP186426 for TUB2). A pathogenicity test was performed to confirm Koch,s postulates. Inoculum was obtained from the two isolates by cultivating in 500 mL of potato dextrose broth on a shaker at 25℃. After 10 days, the hyphae grew into a cluster. The 6 individuals of P. bulbocodioides were divided into two groups. Three individuals grew in the bark substrate containing hyphae cluster, while another 3 individuals grew in the bark substrate containing sterile agar medium. The plants were kept in a greenhouse (constant temperature at 25℃, day and night for 12 h). After 20 days, the group inoculated with F. oxysporum isolates showed the same disease symptoms as observed on the plants in the field, while the control plants remained disease free. F. oxysporum was reisolated from the infected tissues (Sup. S1b, c). Phylogenetic dendrograms of Fusarium oxysporum were grouped by TEF1-α and TUB2 sequence (Sup. S3). The results confirmed that this fungus was identical to those identified by colony morphology, phylogenetic relationship and TEF1-α and TUB2 sequence. To our knowledge, this is the first report of F. oxysporum causing root rot on Pleione species in China. This is a pathogenic fungus in the production of Pleione species. Our study is helpful for the identification of root rot on Pleione species and the development of disease control strategy on cultivation.

First Report of Fusarium oxysporum Causing Stem Rot on Mammillaria humboldtii in China.

Mammillaria humboldtii found in Mexico is a short-globose ornamental cactus species of the Cactaceae family, which has gained increasing popularity in China. It is characterized by tuberculate stems, dimorphic areoles, small pink flowers and pitted seed cell walls. The populations of wild M. humboldtii are critically endangered and are now of international conservation concern. In July 2021, stem rot symptoms were observed on M. humboldtii in a commercial greenhouse located in Zhangzhou (117°39'44.0064″E, 24°28'3.7236″N), Fujian Province (southern China). The typical symptoms were water-soaking, rotting and wilting on the stem, eventually leading to necrosis of the plants within 20 to 30 days. The vascular system of infected stems and roots showed a reddish-brown discolouration. The disease affected approximately 10% of 1000 plants. Fungi were isolated from the diseased stems of 26 samples, which were chopped into small pieces (5 × 5 mm), surface-sterilized with 75% ethanol for 40 s, and placed onto potato dextrose agar (PDA). After seven days of dark culture at 28°C, morphologically similar fungal isolates with whitish aerial mycelium and purple pigment were observed. On carnation leaf agar (CLA), isolates produced sickle and slightly curved macroconidia with three to four septa, measuring 12.8 to 27.9 × 1.9 to 3.8 μm (n = 15), and unicellular, ovoid to elliptical microconidia measuring 3.8 to 7.7 × 1.4 to 2.5 μm (n = 30). Smooth walled chlamydospores were terminal or intercalary, single or in pairs, measuring 9.2 to 13.1 μm (n = 15) in diameter. For molecular identification, the internal transcribed spacer (ITS) region of rDNA (Schoch et al. 2012), translation elongation factor-1α (EF1-α) (Maryani et al. 2019) and gene coding endopolygalacturonase 1 (PG1) (Hirano et al. 2006) of the representative isolate FJMH7 were amplified, purified and sequenced. BLASTn analysis of the ITS, EF1-α and pg1 sequences (GenBank accession numbers: ON832660, ON843495 and ON843496) showed 100%, 99.70% and 98.96% identity with F. oxysporum (GenBank accession numbers: KX611626, OM801797 and KF437345), respectively. Phylogenetic analysis based on the the concatenated ITS and EF1-α sequences and pg1 genes placed isolate FJMH7 with F. oxysporum reference strains in the phylogenetic trees. Based on morphological identification and sequence analysis, this isolate was identified as F. oxysporum. For the pathogenicity assay, six 6-month-old healthy plants of Mammillaria humboldtii were inoculated by dipping roots in a conidial suspension (106 conidia/mL) of isolate FJMH7 cultured in Bilai's medium for three days. Six noninoculated plants treated with Bilai's medium served as a control. Plants were transplanted into pots filled with sterilized soils and placed in a glasshouse at 25°C. After 15 days, all the inoculated plants exhibited rot symptoms on stems, which were similar to those observed in the commercial greenhouses. All inoculated plants were dead 30 days after inoculation. Control plants did not show any symptoms. F. oxysporum was reisolated and confirmed based on morphology and sequencing. No fungi were reisolated from control plants. To fulfil Koch's postulates, the pathogenicity assay was repeated twice with the same results. To date, F. oxysporum isolates have been reported on golden barrel cactus (Echinocactus grusonii) (Polizzi et al. 2004), night-blooming cereus (Hylocereus undatus) (Wright et al. 2007), apple cactus (Cereus peruvianus monstruosus) (Garibaldi et al. 2011), Schlumbergera truncate (Lops et al. 2013), Astrophytum ornatum (Quezada-Salinas et al. 2017) and Nopalea cochenillifera (Santiago et al. 2018). To our knowledge, this is the first report of F. oxysporum on M. humboldtii in China, indicating that this pathogen could cause wilt and rot disease on different cactus hosts.

Entomopathogenic fungi associated with cultivated honeybush, Cyclopia spp., in South Africa and their pathogenicity towards a leafhopper pest, Molopopterus sp. (Hemiptera: Cicadellidae)

The southern and eastern parts of the African Fynbos region favour the production of honeybush tea. Honeybush biomass and extracts are used to prepare a beverage both locally and internationally, mainly as herbal tea with health benefits. Honeybush tea is mostly grown organically requiring natural control measures for pests and diseases. The leafhopper, Molopopterus sp., is one of the most important pests of cultivated honeybush in South Africa, as its feeding compromises the quality and quantity of the yield through leaf discolouration and reduction of the photosynthetic area. Local entomopathogenic fungi (EPF) can provide a pool of potential biocontrol agents for this pest. Therefore, a total of 98 soil samples were collected from organically grown honeybush fields and vegetation surrounding the honeybush fields in the Western Cape province of South Africa. Entomopathogenic fungi were isolated using the insect bait method and were characterised using molecular techniques. Twenty fungal isolates of Metarhizium anisopliae and Fusarium oxysporum were recovered from soil samples, of which 70% were from honeybush fields and 30% were from surrounding vegetation. Fusarium oxysporum isolates comprised 20% of the recovered isolates; M. anisopliae the remainder. Laboratory bioassays of the recovered isolates against adults and nymphs of the leafhopper, showed that F. oxysporum isolates caused 10–45% mortality and M. anisopliae isolates 30–80% mortality. Metarhizium anisopliae isolates J S1, KF S3, KF S11, KF S13, LS1 and LS2 were the most virulent and induced over 60% mortality in both nymphs and adults at a concentration of 1 × 107 conidia/ml.