Wilt Symptoms Research Articles

Bacillus sp. A8a reduces leaf wilting by Phytophthora and modifies tannin accumulation in avocado

The objective was to assess the biocontrol capacity of Bacillus sp. A8a in avocado (Persea americana) plants infected by Phytophthora cinnamomi. A greenhouse experiment was implemented with four treatments: 1) control plants; 2) plants infected with P. cinnamomi; 3) plants inoculated with Bacillus sp. A8a; 4) plants infected with P. cinnamomi and inoculated with Bacillus sp. A8a. We evaluated several morpho-physiological variables during the experiment, which lasted 25 days after infection (dai). Moreover, we analyzed tannin density in stems at 25 dai to determine the plant defense response against the disease. Inoculation with strain A8a reduced wilting symptoms by 49 % at 25 dai, compared with non-inoculated plants. No differences were detected in morphophysiological variables between treatments. However, a greater tannin accumulation was registered in the xylem of infected plants, whilst plants inoculated with strain A8a displayed a larger tannin density in the cortex. Our results confirm the biocontrol activity of Bacillus sp. A8a in avocado plants and suggest that tannin differential accumulation in the cortex of plants inoculated with the bacteria may contribute to the enhanced tolerance of avocado plants against Phytophthora root rot.

First specific detection and validation of tomato wilt caused by Fusarium brachygibbosum using a PCR assay.

Tomato wilt is a widespread soilborne disease of tomato that has caused significant yield losses in many tomato growing regions of the world. Previously, it was reported that tomato wilt can be caused by many pathogens, such as Fusarium oxysporum, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Fusarium acuminatum, and Plectosphaerella cucumerina. In addition, we have already reported that Fusarium brachygibbosum caused symptomatic disease of tomato wilt for the first time in China. The symptoms of tomato wilt caused by these pathogens are similar, making it difficult to distinguish them in the field. However, F. brachygibbosum specific identification method has not been reported. Therefore, it is of great importance to develop a rapid and reliable diagnostic method for Fusarium brachygibbosum to establish a more effective plan to control the disease. In this study, we designed F. brachygibbosum-specific forward primers and reverse primers with a fragment size of 283bp located in the gene encoding carbamoyl phosphate synthase arginine-specific large chain by whole genome sequence comparison analysis of the genomes of eight Fusarium spp.. We then tested different dNTP, Mg2+ concentrations, and annealing temperatures to determine the optimal parameters for the PCR system. We evaluated the specificity, sensitivity and stability of the PCR system based on the optimized reaction system and conditions. The PCR system can specifically identify the target pathogens from different fungal pathogens, and the lower detection limit of the target pathogens is at concentrations of 10 pg/uL. In addition, we can accurately identify F. brachygibbosum in tomato samples using the optimized PCR method. These results prove that the PCR method developed in this study can accurately identify and diagnose F. brachygibbosum.

Identification and characterization of Fusarium falciforme, incitant of wilt disease in cashew seedlings and its management

The present study was carried out to identify and characterize the pathogen responsible for cashew seedling wilt, and to find its control measures. Characteristic symptoms of wilt included yellowing and drying of the lower leaves at the initial stage. In the advanced stage, drooping and wilting of the top portion of the seedling was more common. Pathogen Fusarium falciforme, was identified based on the morphological characteristics and ITS sequence data. Several fungicidal formulations effectively reduced the intensity and severity of seedling wilt disease. Phylogenetic analysis placed all the five isolates into a single cluster. A mixture of Trifloxystrobin and Tebuconazole inhibited the pathogen in terms of mycelial growth, followed by Carbendazim + Mancozeb with ED50 values of 4.370 and 8.427 ppm, respectively. However, mixture of Carbendazim and Mancozeb was found the best suitable fungicide formulation to reduce the severity of wilt disease and the highest percent disease control over the untreated one.

The SIX5 Protein in Fusarium oxysporum f. sp. cepae Acts as an Avirulence Effector toward Shallot (Allium cepa L. Aggregatum Group).

Fusarium oxysporum f. sp. cepae (Foc) causes basal rot disease in Allium species, including onions (Allium cepa L.) and shallots (A. cepa L. Aggregatum group). Among Allium species, shallots can be crossbred with onions and are relatively more resistant to Foc than onions. Thus, shallots are considered a potential disease-resistant resource for onions. However, the mechanisms underlying the molecular interactions between shallots and Foc remain unclear. This study demonstrated that SIX5, an effector derived from Foc (FocSIX5), acts as an avirulence effector in shallots. We achieved this by generating a FocSIX5 gene knockout mutant in Foc, for which experiments which revealed that it caused more severe wilt symptoms in Foc-resistant shallots than the wild-type Foc and FocSIX5 gene complementation mutants. Moreover, we demonstrated that a single amino acid substitution (R67K) in FocSIX5 was insufficient to overcome shallot resistance to Foc.

First report of Fusarium wilt of Cavendish bananas caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 in the Grande Comoros Island.

Fusarium wilt of banana, caused by the soil-borne pathogen Fusarium oxysporum f. sp. cubense (Foc), is a major constraint to banana production worldwide (Viljoen et al., 2020). Currently, Cavendish bananas are severely affected by Foc Tropical Race 4 (TR4) globally. In Africa, Foc TR4 was first detected in northern Mozambique in 2013 (Viljoen et al., 2020), and has since been found on the island of Mayotte in the Mozambique Channel off the coast of southeastern Africa (Aguayo et al., 2021). In early 2023, severe leaf-yellowing and wilting of Cavendish banana plants was observed on Cavendish banana plants at several small holder farmer properties in Grande Comoros (Ngazidja) including in Ntsinimoipanga (-11,790054 S, 43°25'47,04384 E), Batou (-11,499716 S, 43°21'51,71976''E), Madjeweni (-11,8217 S , 43°16'41''E) and Mdé (-11°41'54'' S, 43°15'20''E). When the pseudostems of these plants were split open, a reddish-brown internal discoloration of the vascular tissue became apparent. Discolored strands of diseased plants were collected, and the causal agent identified using DNA-based techniques, vegetative compatibility group (VCG) analysis and pathogenicity testing. The samples were plated onto potato dextrose agar and single-spored and isolated from individual diseased plants identified as F. oxysporum based on cultural and morphological characteristics. These include the production of white fungal colonies with a purple center, infrequent production of macroconidia, but an abundance of microconidia on short monophialides, and terminal or intercalary chlamydospores (Leslie and Summerell, 2006). Foc TR4 was identified from seven isolates by conventional (Dita et al., 2010) and quantitative-PCR (Matthews et al., 2020), and with loop-mediated isothermal amplification (LAMP) (Ordóñez et al., 2021). All seven isolates were confirmed as members of the VCG 01213/16 complex when nit-1 mutants of the unknown Foc isolates were compatible with Nit-M mutants of the Foc VCG 01213 and VCG 01216 tester strains. Two isolates were then selected for pathogenicity testing, and 2-month-old tissue culture-derived Cavendish plants (cv. Williams) inoculated by using the method described by Ndayihanzamaso et al. (2022). After 4 weeks, the Foc TR4-inoculated plants produced wilting symptoms and internal rhizome discoloration typical of Fusarium wilt. Foc TR4 was reisolated from the inoculated plants and identified by qPCR (Matthews et al. 2020), thereby fulfilling Koch's postulates. These results provide scientific proof of the presence of Foc TR4 in a second island in the Comoros archipelago. Comprehensive surveys will be conducted in all three of the Comoros Islands to assess the presence and impact of Foc TR4 to implement containment strategies. Collaborative initiatives and coordinated actions among growers and other stakeholders are needed to prevent the spread of Foc TR4 to more Southwest Indian Ocean islands and countries on the East African coasts. The importance of banana for food security and livelihoods, and the unique genetic diversity of bananas found on the Comoros islands, requires the eradication and isolation of diseased bananas on the short term, and the screening of local banana varieties for Foc TR4 resistance on the longer term.

Recognising ceratocystis disease symptoms in a Eucalyptus pellita nursery

Early recognition of symptoms of infection is essential to prevent diseases from spreading through nurseries and into planted systems. In plantations, symptoms of Ceratocystis manginecans infection are typically wilt and canker but symptoms may differ at the nursery stage and as yet have not been described for Eucalyptus pellita. In this study, disease symptoms were recorded over a three-month period in an E. pellita nursery and isolates trapped. Then, to clearly identify those relating to ceratocystis disease and any related mortality, two C. manginecans isolates were re-inoculated by either spraying or dipping of healthy plantlets raised by tissue culture and mini-cuttings. Many disease symptoms were observed in the nursery but only perithecia on the leaves and stems was a clear indication of the presence of a Ceratocystis pathogen. Nine pathogenic fungal species, including C. manginecans, were isolated and identified by DNA analysis. Koch’s postulates indicated that leaf blight associated with C. manginecans was black-brown in colour, irregular, with uneven edges and surrounded by a yellow margin or halo on the upper leaf surface. Mortality following inoculation of plantlets by spraying immediately after planting was > 70%; by contrast, mortality of mini-cuttings was < 50% if spraying was done just before transplanting but < 20% if done 7 or 14 days after transplanting. Isolate EP313C caused significantly greater mortality than isolate EP106C. The research showed that C. manginecans and other pathogens were present in the nursery environment and must be identified and managed as part of good nursery practice.

Priming corn resistance to Goss’s wilt using a weakly aggressive strain of Clavibacter nebraskensis

Goss’s wilt, caused by the gram-positive bacterium Clavibacter nebraskensis, has become one of the most widely spread corn diseases in the USA and Canada. Considered as a relatively new disease, with limited knowledge about the more recently renamed pathogen causing it is, it is today a serious yield-limiting factor in corn crops in North America. The diversity of C. nebraskensis strains translates into the differential plant responses it causes, ranging from hypersensitive-like response to partial resistance, or otherwise susceptibility expressed as dark, water-soaked spots and shiny patches of dried bacterial ooze on the lesions. Weakly aggressive strains of C. nebraskensis typically appear as the season winds down and the crop is about to be harvested, whereas highly aggressive strains can be found at any point during the growing season. Previous research showed that plant’s ethylene and reactive oxygen species (ROS) are essential in limiting the spread of Goss's wilt symptoms near infection sites by triggering programmed cell death (PCD). Preliminary observations that certain strains induce strong defense responses with very little to no symptoms fed the idea to use them for disease resistance priming. The induction of biosynthetic or responsive genes of ethylene, ROS, and salicylic acid (SA) both at the local and distal tissues is in line with the limited disease symptoms development in response to priming with the weakly aggressive strain before further inoculation with a highly aggressive one. The results further expand our understanding of differential host responses to strains with different levels of aggressiveness. The priming process led to an inhibition of the elongated lesions in the susceptible corn plants in comparison with the unprimed plants. The expression patters of a lectin receptor-like kinase, Salt Intolerance 2 (ZmSIT2), and blue copper binding protein (ZmBCP) suggests them as key mediators of ethylene and ROS homeostasis in corn tissues upon infection. This adds to the pool of knowledge on corn defenses against bacterial pathogens and the mechanism of its biopriming, which could serve as an environment-friendly alternative method to control Goss’s wilt.

First report of Ceratocystis ficicola causing wilt disease on common fig (Ficus carica) in Italy.

Common fig (Ficus carica L.) in is one of the most important crops in the Mediterranean area. In Italy, it is grown on a total area of 2118 ha. In Apulia (South-eastern Italy), the annual production of fig exceeds 3200 tons annually and together with olive and grapevine, they characterize the Apulian agricultural panorama. In September 2021, symptoms of a vascular wilt disease and, in severe cases, decline tree mortality were observed in Salento area (Apulia). Symptomatic Affected plants showed symptoms of leaf wilt and different stages of disease expression, which begins with leaf chlorosis on shoots, followed by wilting, extensive defoliation and twig dieback. On the main branches andlower part of the trunk and in some cases on lateral branches, bark cracks and cankers were observed and extended wood discoloration was detected in cross sections. In two orchards located in Salice Salentino (Site 1) and Squinzano (Site 2), where disease incidence exceeded 80%, 3-5 wood discs per tree were gathered from affected tissues from two (Site 1) and four (Site 2) trees. Isolations were performed on malt extract agar 2% amended with 0.5 g L-1 streptomycin sulfate. A Ceratocystis species was recovered from all samples trees with high frequencies (83.3%). Two-week-old colonies on potato dextrose agar showed black ascomata with 300-600 µm wide bases and 1100-2250 µm long necks. On the tips of the necks, ascospores (5-6x4-5 µm) exuded in creamy white sticky masses. Endoconidia (5-9x4.5-7 µm) were abundant, cylindrical, aseptate, and produced in chains. Two monoconidial representative strains CRSFA.Cer.033 (Site 1) and CRSFA.Cer.035 (Site 2) were deposited in the DISSPA Di.S.S.P.A. collection of the University of Bari. Species identification was done through sequence analyses of rDNA internal transcribed spacer region (ITS) using ITS5/ITS4 primers (White et al. 1990), elongation factor 1 alpha gene (TEF) using EF1/EF2 (O'Donnell et al. 1998) and RNA polymerase II gene (RPB2) gene using RPB2-5F/FRPB2fRPB2-7cR (Liu et al. 1999). Sequences were deposited in NCBI GenBank (accession numbers: OQ329983-OQ335969 (ITS), OQ352265-OQ352266 (TEF), OQ352268-OQ352267 (RPB2)). The sequences of both Apulian isolates were identical. BLAST searches revealed high similarity to the sequences of two isolates of Ceratocystis ficicola Kajitani and Masuya from Japan: ex-type CMW38543 and CMW38544, specifically 98.41% identity matching with KY685076 (ITS), 100% with KY685079 (TEF), and 99.87% with KY685083 and KY685082 (RPB2). Pathogenicity tests were conducted, on six common fig 'Dottato' seedlings by inoculating one year-old twigs with mycelium plugs (Bolboli et al. 2022). Control plants were inoculated with PDA plugs without mycelium. After one month, all inoculated twigs showed symptoms of wilt. Forty days post-inoculation, the bark of inoculated twigs was removed, and longitudinal and transverse sections revealed wood discolorations extending above and below the inoculation point. Ceratocystis ficicola was consistently reisolated from symptomatic tissues and identified as described above, thus fulfilling Koch's postulates. Neither symptoms nor positive isolations were observed in control seedlings. The pathogen was first described in 2011 in Japan (Kajitani and Masuya, 2011) where it currently affects all fig-producing areas. In 2018, it was reported for the first time in the EPPO region in Greece (Tsopelas et al., 2021), and to our knowledge, this is the first report on its detection in Italy. Since February 2022, C. ficicola has been included in the EPPO alert list because of its potential to cause tree mortality and the difficulty of its eradication. Regional national surveys are therefore urgently needed to determine its distribution in the fig growing areas of Italy and limit its spread. References Bolboli, Z., et al. 2022. Mycol. Prog. 21:89. doi: 10.1007/s11557-022-01834-9 Kajitani, Y., and Masuya, H., 2011. Mycoscience 52:349. doi: 10.1007/s10267-011-0116-5 Liu, Y. J., et al. 1999. Mol. Biol. and Evol. 16:1799. doi: 10.1093/oxfordjournals.molbev.a026092 O'Donnell, K., et al. 1998. Proc Natl Acad Sci USA 95:2044. doi: 10.1073/pnas.95.5.2044 Tsopelas, P., et al. 2021. Phytopathol. Mediterr. 60:337. doi: 10.36253/phyto-12794 White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. doi: 10.1016/0307-4412(91)90165-5.

Reevaluation of the Application Method and Efficacy of Propiconazole in Controlling Laurel Wilt in Avocado Orchards in South Florida

Propiconazole has fungistatic and fungicidal properties against Harringtonia lauricola, the causal agent of laurel wilt disease. Propiconazole injections are used by Florida, USA, farmers as a prophylactic method to manage the disease in avocado (Persea americana) trees, but its efficacy has remained questionable for more than a decade due to documented restricted mobility within the tree vascular system. This study was conducted to evaluate the absorption of propiconazole when using soil drenching as an alternative application method, assess the efficacy of propiconazole in controlling disease development when drenched or injected, and its synergistic effect on common cultural management practices used by the local farmers, such as branch removal and trunk cutting (“stumping”). To determine if propiconazole soil-drenching can provide better xylem coverage, potted and mature orchard trees were treated with different concentrations and artificially inoculated with the pathogen. Propiconazole translocation from the roots to above-the-ground tissue was confirmed in potted and orchard trees, but the concentrations in orchard trees were below the fungicidal threshold (1 ppm). Although none of the potted trees developed laurel wilt symptoms, all inoculated branches of the orchard trees did. Furthermore, noninoculated branches in more than 80% of the inoculated and propiconazole-treated orchard trees developed symptoms, even though the inoculated branch was cut at the early stages of disease development. To elucidate if propiconazole injections effectively control the disease, trees from a commercial orchard that were injected five times were challenged by artificial inoculation. Propiconazole concentration in trees was highly variable (ranging from &lt; 0.01 to 294 ppm), but even in trees with a high concentration of propiconazole, inoculated and noninoculated branches developed symptoms. Even though drenched and injected trees were “stumped” soon after symptoms appeared in the noninoculated branches (4 to 5 months after inoculation), all of the stumps in the drenched plot and 80% of the injected trees, showed internal symptoms 5 and 4 months after the cut, respectively. Results demonstrate that the soil-drenching of propiconazole is an ineffective application method in orchard trees, and that the conventional injection does not prevent disease development after artificial inoculation. Moreover, because propiconazole does not prevent the movement of the pathogen to the trunk, the “stumping” of infected trees to reduce the disease in the orchard is an inadequate practice. This study highlights the critical need for other active ingredients with lower fungicidal thresholds, longer half-life, and higher xylem mobility.

First Report of Fusarium oxysporum Causing Leaf Wilt on Dinteranthus vanzylii (Green Stone Plant) in China.

Dinteranthus vanzylii is a low-growing species in the family Aizoaceae, native to southern Africa, with a pair of thick grey leaves covered with dark red spots and stripes. This stone-like succulent grows near the ground, which may protect it from water evaporation and herbivores. Dinteranthus vanzylii has become popular in China due to its attractive appearance and easy indoor cultivation. In September 2021, 7% of D. vanzylii (approximately 140 pots) showed leaf wilt symptoms in a commercial greenhouse located in Ningde (119°35'39.696″E, 27°23'30.556″N), Fujian Province, China. The diseased plants were shrivelling and eventually underwent necrosis. Their leaf tissues were rotting and carpeted with white mycelium. The leaf tissues of 10 symptomatic plants were cut into 0.5 cm2 pieces, surface-sterilized and placed on PDA medium. According to the colony morphology after 7 days of culture, 20 fungal isolates with abundant whitish aerial mycelium were divided into two types: 8 isolates produced lilac pigment whereas 12 did not. Both produced unicellular ovoid microconidia, sickled-shaped macroconidia with 3 - 4 septa and single or paired smooth, thick-walled chlamydospores on carnation leaf agar (CLA). Molecular identification based on DNA sequences from EF1-α (O'Donnell et al. 1998), RPB1 and RPB2 (O'Donnell et al. 2010) revealed 100% identity among isolates within each group; however, there were several base differences between two types. Sequences of representative isolates KMDV1 and KMDV2 were deposited in GenBank (acc. nos.: OP910243, OP910244, OR030448, OR030449, OR030450 and OR030451), which showed 99.10% - 99.74% identity with different F. oxysporum strains (GenBank acc. nos.: KU738441, LN828039, MN457050, MN457049, ON316742 and ON316741). Phylogenetic tree inferred from the concatenated EF1-α, RPB1 and RPB2 revealed that these isolates clustered with F. oxysporum. Thus, these isolates were identified as F. oxysporum. Using a root-drenching method, 10 one-year-old healthy D. vanzylii were inoculated in conidial suspensions (1*106 conidia/mL) of isolates KMDV1 and KMDV2 for 60 min, respectively. They were transplanted into pots with sterilized soil and incubated in a plant-growth chamber at 25°C and 60% relative humidity. Control plants were treated with sterilized water. The pathogenicity test was repeated three times. All plants inoculated with each isolate developed leaf wilt symptoms after 15 days and were dead after 20 - 30 days. However, no symptoms were observed in the control plants. Fusarium oxysporum was reisolated and confirmed based on morphology and EF1-α sequence analysis. No pathogens were isolated from the control plants. This is the first report of F. oxysporum causing leaf wilt disease on D. vanzylii in China. To date, several diseases have been reported on members of the Aizoaceae. For instance, collar and stem rot on Lampranthus sp. caused by Pythium aphanidermatum (Garibaldi et al. 2009), wilt on Lampranthus sp. and Tetragonia tetragonioides caused by Verticillium dahliae (Garibaldi et al. 2010; Garibaldi et al. 2013), and leaf spot on Sesuvium portulacastrum caused by Gibbago trianthemae (Chen et al., 2022). Our research could provide insight into fungal diseases on members of the Aizoaceae and contribute to their cultivation and management.

First report of Fusarium wilt of Cavendish bananas caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 in Venezuela.

Fusarium wilt of banana (Musa spp.), caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is a major constraint to banana production worldwide (Dita et al., 2018). A strain of Foc that affects Cavendish (AAA) bananas in the tropics, called Foc tropical race 4 (TR4; VCG 01213), is of particular concern. Foc TR4 was first detected in Malaysia and Indonesia around 1990 but was restricted to Southeast Asia and northern Australia until 2012. The fungus has since been reported from Africa, the Indian subcontinent, and the Middle East (Viljoen et al., 2020). Foc TR4 was detected in Colombia in 2019 and in Perú in 2021 (Reyes-Herrera et al., 2020). The incursions into Latin America and the Caribbean (LAC) triggered global concerns, as 75% of international export bananas are produced in the region. Banana production in Venezuela, however, is primarily intended for domestic consumption (Aular and Casares, 2011). In 2021 the country produced 533,190 metric tons of banana on an area of 35,896 ha, with an approximate yield of 14,853 kg/ha (FAOSTAT, 2023). In July 2022, severe leaf-yellowing, and wilting, along with internal vascular discoloration of the pseudostem, were noted in Cavendish banana plants cultivar 'Valery' in the states of Aragua (10°11'8″N; 67°34'51″W), Carabobo (10º14'24″N; 67º48'51″W), and Cojedes (9°37'44″N; 68°55'4″W). Necrotic strands from the pseudostems of diseased plants were collected for identification of the causal agent using DNA-based techniques, vegetative compatibility group (VCG) analysis and pathogenicity testing. The samples were first surface disinfected and plated onto potato dextrose agar medium. Single-spored isolates were identified as F. oxysporum based on cultural and morphological characteristics, including white colonies with purple centres, infrequent macroconidia, abundant microconidia on short monophialides, and terminal or intercalary chlamydospores (Leslie and Summerell, 2006). Foc TR4 was identified from five isolates by endpoint and quantitative-PCR using four different primer sets (Li et al. 2013; Dita et al. 2010; Aguayo et al. 2017; Matthews et al. 2020). The same isolates were identified as VCG 01213 by successfully pairing nitrate non-utilizing (nit-1) mutants of the unknown strains with Nit-M testers of Foc TR4 available at Stellenbosch University (Leslie and Summerell, 2006). For pathogenicity testing, 3-month-old Cavendish banana plants cultivar 'Williams' were inoculated with isolates from Venezuela grown on sterile millet seed (Viljoen et al., 2017). Plants developed typical Fusarium wilt symptoms 60 days after inoculation, including yellowing of leaves that progressed from the older to the younger leaves, wilting, and internal discoloration of the pseudostem. Koch's postulates were fulfilled by reisolating and identifying Foc TR4 from the plants by qPCR (Matthews et al., 2020). These results provide scientific proof of the presence of Foc TR4 in Venezuela. The Venezuelan Plant Protection Organization (INSAI) has declared Foc TR4 as a newly introduced pest (January 19, 2023), and infested banana fields were placed under quarantine. Comprehensive surveys are now conducted in all production areas in Venezuela to assess the presence and impact of Foc TR4, and information campaigns were started to make farmers aware of biosecurity protocols. Collaborative initiatives and coordinated actions among all stakeholders are needed to prevent the spread of Foc TR4 to other countries in Latin America, and to develop Foc TR4-resistant bananas (Figueiredo et al. 2023).

Responses of common enset (Ensete ventricosum) varieties in Ethiopia to Xanthomonas and virulence of Xanthomonas strains

Enset is a vital food crop for over 20% of the population living in Ethiopia. Enset bacterial wilt (EBW) disease caused by a pathogen Xanthomonas vasicola pv. Musacearum (Xvm) is among the challenges for its production. This study evaluated the tolerance level of enset varieties in Gamo highland against Xvm. During the field survey, EBW infected enset samples were collected for isolating and characterizing Xanthomonas responsible for causing EBW disease. The isolates were used for infecting different enset varieties in the field. Soil samples were also collected from the farm before enset cultivation (F-BC), during enset cultivation (F-DC), and after EBW disease development (F-AD). Among 126 bacterial isolates, 18 Xvm were found to yield amplicons at 420 bp using Xcm-36 as a specific primer. During pathogenicity tests, all Xvm isolates caused EBW disease before 38 days in the Bodha enset variety. Except for the variety Maze, all the field-tested varieties began to show a typical wilt symptom after 45 days of infected enset cultivation. Isolate X-D5 showed a significantly (P ≤ 0.05) higher virulent capacity to all enset varieties tested than isolates X–B4 and X–K4. Overall, findings of the fieldwork provide clues on the traditional knowledge of characterizing different enset varieties for susceptibility and tolerance to Xvm that can be useful in designing EBW disease management strategies.

Identification of Strawberry Wilt Caused by Plectosphaerella cucumerina in China.

Strawberry (Fragaria × ananassa Duch.) is a widely cultivated economic crop in China. In April 2022, an unusual wilt disease was observed on strawberry plants (6 months' old) in Chenzui town, Wuqing district, Tianjin, China (117°1'E, 39°17'N). The incidence across the greenhouses (≈0.34 ha) was approximately 50 to 75%. The first wilt symptoms were observed on the outer leaves, then the whole seedlings wilted and died. The rhizome of the diseased seedlings changed color and became necrotic and rotted. Symptomatic roots were surface disinfected with 75% ethanol for 30 s, washed with sterile distilled water for three times, and then cut into 3 mm2 pieces (four pieces per seedling) and placed on petri dish with potato dextrose agar (PDA) containing 50 mg/L of streptomycin sulfate and incubated at 26℃ in the dark. After 6 days' incubation, hyphal tips of the growing colonies were transferred onto PDA. Eighty-four isolates belong to five fungal species were obtained from 20 diseased root samples based on their morphological characteristics. To confirm their pathogenicity, ten two-month-old healthy seedlings of strawberry (cv Red Face) planted in sterilized nutrient soil, were inoculated by pouring 50 mL of conidial suspension (107 conidia/mL) (Cai et al. 2021). Another ten seedlings poured with sterile distilled water were used as controls. Each treatment was repeated three times in a greenhouse at 25 to 28℃ and 75% relative humidity under a 12-h photoperiod. After 15 days，only seedlings inoculated with Plectosphaerella (an original percentage of 35.71%) exhibited similar symptoms to those of diseased seedlings originally observed in the field. Seedlings had no symptoms in the control and other fungi inoculation treatments. To fulfill the Koch's postulates, Plectosphaerella isolates were reisolated from each inoculated, symptomatic seedling with a percentage of 100%, but were not recovered from any of the control seedlings. The experiments were repeated twice with similar results. The results indicated that the genus Plectosphaerella was the pathogen causing strawberry wilt. Colonies of the genus Plectosphaerella isolates on PDA were white to cream, and then gradually became salmon pink, with few aerial hyphae and slimy surfaces. Colonies produced numerous hyphal coils with conidiophores. Conidia were 4.56 to 10.07 μm × 1.11 to 4.54 μm (avg. 7.10 × 2.56 μm, n=100), septate or aseptate, ellipsoidal, hyaline and smooth. Such morphological characteristics were identical to those of Plectosphaerella spp. (Palm et al. 1995). For species identification, the ITS region and D1/D2 domain of the 28S rRNA gene of representative isolates (CM2, CM3, CM4, CM5 and CM6) were amplified and sequenced with the primer pair ITS1/ITS4 and NL1/NL4, respectively (White et al. 1990; O'Donnell and Gray 1993). By BLASTn analysis, the obtained sequences of ITS amplicon (ON629742, ON629743, ON629744, ON629745, and ON629746) and D1/D2 domain amplicon (OQ519896, OQ519897, OQ519898, OQ519899, and OQ519900) showed 99.14% to 99.81% identity to sequences of P. cucumerina (MW320463.1 and HQ239025.1) in the NCBI database. A multilocus phylogenetic tree performed by the UPGMA analysis showed that the representative isolates were assigned to the group of P. cucumerina. To our knowledge, this is the first report of P. cucumerina causing strawberry wilt worldwide. This disease may induce serious economic losses in strawberry production, thus effective management strategies should be taken.

First Report of Hemp Fusarium Wilt Caused by Fusarium oxysporum in Croatia.

Wilted hemp (Cannabis sativa L.) plants were observed in August 2019 in commercial fields around Osijek, Croatia. Plants and roots with disease symptoms were collected. The single-spored isolates produced septate cottony white to light pink aerial mycelium and purple undersurface on potato dextrose agar (PDA). Smooth and hyaline hyphae were branched and septate. Macroconidia were fusiform to sickle-shaped with foot-shaped basal cells, elongated apical cells and three to five septa. Sequencing of the internal transcribed spacer and the partial elongation factor 1-α gene identified the species as Fusarium oxysporum. Artificial infection fulfills Koch's postulates, producing plants which show stunted growth and wilt symptoms similar to those observed in the commercial fields. Control seedlings remained symptomless and healthy. To the best of our knowledge, this is the first report of hemp Fusarium wilt causing F. oxysporum in Croatia. Considering that F. oxysporum has been reported in main field crops in Croatia, the presence of this pathogen could cause economically significant hemp production decreases, especially in humid and cold springs and susceptible varieties.

Identifying F. oxysporum Strains Causing Wilt in Southern Indian Chickpeas

Fusarium oxysporum (Schletend: Fr) f. sp. ciceri (Padwick), referred to as Foc, is a soil-borne fungus that poses a constant threat to chickpeas (Cicer arietinum L.) by causing wilt disease. Typical wilt symptoms were observed in chickpea plants collected from 24 different locations across three southern Indian states. The process involved isolating Fusarium species from the roots of these wilted plants, resulting in the identification of various strains exhibiting diverse cultural and morphological characteristics on potato dextrose agar medium. All twenty four isolates were subjected to Koch's postulates using the standard method, which yielded varied responses in terms of disease incidence. After analyzing cultural, morphological, molecular traits, and conducting pathogenicity tests, the fungus was definitively identified as F. oxysporum Schlechtend. Fr. f. sp. ciceri (Padwick) Matuo and K. Sato. Among the 24 isolates tested on the chickpea wilt susceptible cultivar JG-62, one was non-pathogenic with zero percent disease incidence (PDI), while one isolate was highly pathogenic showed 100 percent PDI. Highly pathogenic four isolate was further used for molecular identification with secreted in xylem primers (SIX). Comparative studies of cultural traits and conidial morphology among different isolates revealed variations in growth patterns, pigmentation, sporulation, and the size and structure of macro and micro conidia, as well as chlamydospores.

Optimization of the Use of Industrial Wastes in Anaerobic Soil Disinfestation for the Control of Fusarium Wilt in Strawberry.

Anaerobic soil disinfestation (ASD) is proposed as an alternative to the use of chemical fumigants against Fusarium wilt in strawberry crops. Different residual wastes (rice bran, fishmeal, and residual strawberry extrudate) were assessed as amendments for ASD. Two different concentrations and two incubation durations were tested in growth chamber trials. The abundance of several microbial groups was noted before and after the treatments. Strawberry plants were grown in the treated soils to record Fusarium wilt disease severity. The population density of F. oxysporum increased after ASD in most amendments with rice bran and residual strawberry extrudate. Changes in Trichoderma spp., copiotrophic bacteria, and Streptomyces spp. populations were observed after anaerobiosis treatments and plant trials. A reduction in the disease severity was achieved in ASD-treated soils with 20 t/ha of rice bran at both 25 and 60 days of incubation, but not when using a 13.5 t/ha dose. Similarly, treatments using 19.3 t/ha of fishmeal for both incubation durations were able to reduce disease severity. In contrast, a severity reduction was only obtained in soils treated with 25.02 t/ha of the residual strawberry extrudate and incubated for 60 days in anaerobic conditions. Two of the three by-products tested were able to reduce Fusarium wilt symptoms in strawberry plants after an ASD-treatment period of only 25 days. Accordingly, the technique seems promising for strawberry growers in Huelva, Spain, and highly sustainable by giving value to residues produced in surrounding areas.

First report of tomato wilt caused by Fusarium brachygibbosum in China.

Tomato (Solanum lycopersicum) is a common vegetable in the Solanaceae family, which as a nutritious vegetable, is widely planted in China. In July 2022, typical wilt symptoms were observed in tomato fields located in the regions of Shiyan (31.343781°N, 110.901005°E), Hubei. Surveys were performed on tomato plants showing symptoms of leaf chlorosis, dry wilt, and vascular wilts on stem and root. The disease incidence ranged from 40% to 70% in 12 surveyed fields, 11.2 ha in total.. After using a sterilized scalpel to cut a small piece of diseased tomato stem and root tissue, diseased tissue was surface disinfested in 75% ethanol for 30s, placed onto potato dextrose agar (PDA) medium,and incubated at 25℃ for 3 days. Then, the emerging single fungal hypha tip was cut and transferred into PDA plates to obtain single spore isolates. Sixteen fungi growing on PDA plates were initially white colonies with abundant aerial mycelium. After seven days of growth, the center of the plate was yellow to orange and red pigmentation was produced. The five-day-old cultures grown on mung bean medium produced macroconidia that were scarce and scattered, three to four septa, wide central cells, slightly sharp apexes, and ranged in size from 12.6-23.6 μm×2.8-4.1 μm (n=30). Microconidia were slightly curved, ovoid with zero to two septa, measuring 5.2-11.8 μm×1.8-2.7μm (n=30). Spherical chlamydospores were terminal or intercalary, and measured a diameter of 8.1-11.6 μm (n =30). Therefore, sixteen isolates were morphologically identified as Fusarium spp. Moreover, the genomic DNA of the isolates (HBSY-1,HBSY-2, and HBSY-3) was extracted for amplification and sequencing of the regions of internal transcribed spacer (ITS) (White et al., 1990), nuclear large subunit rRNA (nLSU) (O'Donnell, 1992; Vilgalys and Hester, 1990), and the translation elongation factor 1-alpha (EF1-α) (O'Donnell et al. 1998) with primers ITS1/ITS4, NL1/LR3, and EF1/2, respectively. Sequences were submitted to GenBank under accession numbers OP959509, OQ568650, OQ568651 (ITS), OQ186731, OQ568652, OQ568653 (nLSU), and OP957576, OQ572485, OQ572486 (EF1-α). BLASTn showed that the sequences ITS, nLSU, and EF1-α were matched 99.61% (508/510 bp; KU528864.1), 99.90% (993/994 bp; GQ505450.1), and 99.85% (651/652; ON032449.1) to Fusarium brachygibbosum, respectively. Multilocus phylogenetic analysis showed that the isolate was of the same clade as F. brachygibbosum. Therefore, morphological characterization and molecular data identified the fungus as F. brachygibbosum. Pathogenicity of the isolate (HBSY-1) was tested on ten seedlings of tomato (cv. Hezuo908). Tomatoes were inoculated by spraying with conidial suspensions (1×107 spores/mL) at the rootstock region of each plant. In addition, ten negative control plants were treated with sterile water. All plants were incubated in an artificial climate box (LongYue, ShangHai) at 25℃ for 12 days. The experiment was repeated three times. Twelve days later, inoculated tomatoes developed typical wilting symptoms of leaves and vascular wilts of stem and root, while the control plants remained healthy. Thus, pathogens were reisolated from the stems of inoculated plants and not from control plants. To our knowledge, this is the first report of F. brachygibbosum causing leaf wilt and vascular wilts of stem and root on tomatoes in China.

First report of Fusarium oxysporum f. sp. fragariae race 2 causing Fusarium wilt of strawberry (Fragaria × ananassa) in California.

In California, Fusarium wilt of strawberry is widespread and causes significant yield losses. Resistant cultivars with the FW1 gene were protected against Fusarium wilt because all strains of Fusarium oxysporum f. sp. fragariae (Fof) in California were race 1 (i.e., avirulent to FW1-resistant cultivars) (Henry et al. 2017; Pincot, et al. 2018; Henry et al. 2021). In the fall of 2022, severe wilt disease was observed in an organic, summer-planted strawberry field in Oxnard, California. Fusarium wilt symptoms were common and included wilted foliage, deformed and highly chlorotic leaflets, and crown discoloration. The field was planted with Portola, a cultivar with the FW1 gene that is resistant to Fof race 1 (Pincot et al. 2018; Henry et al. 2021). Two samples, each consisting of four plants, were collected from two different locations within the field. Crown extracts from each sample were tested for Fof, Macrophomina phaseolina, Verticillium dahliae, and Phytophthora spp. by recombinase polymerase amplification (RPA) (Steele et al. 2022). Petioles were surface sterilized in 1% sodium hypochlorite for 2 minutes and plated on Komada's medium to select for Fusarium spp. (Henry et al. 2021; Komada, 1975). The RPA results were positive for M. phaseolina in one sample and negative for all four pathogens in the other sample. Salmon-colored, fluffy mycelia grew profusely from petioles of both samples. Colony morphology and non-septate, ellipsoidal microconidia (6.0-13 μm × 2.8-4.0 μm) borne on monophialides resembled F. oxysporum. Single hyphal tip isolation of fourteen cultures (P1-P14) was done to purify single genotypes. None of these pure cultures amplified with Fof-specific qPCR (Burkhardt et al. 2019), confirming the negative result obtained with RPA. Translation elongation factor 1-alpha (EF1α) was amplified using EF1/EF2 primers (O'Donnell et al. 1998) from three isolates. Amplicons were sequenced (GenBank OQ183721) and found through BLAST search to have 100% identity with an isolate of Fusarium oxysporum f. sp. melongenae (GenBank FJ985297). There was at least one nucleotide difference when compared to all known strains of Fof race 1 (Henry et al. 2021). Five isolates (P2, P3, P6, P12, and P13) and an Fof race 1 control isolate (GL1315) were tested for pathogenicity on Fronteras (FW1) and Monterey (fw1; susceptible to race 1). Five plants per isolate × cultivar combination were inoculated by dipping roots in 5 × 106 conidia per mL of 0.1% water agar, or in sterile 0.1% water agar for the negative control, and grown as described by Jenner and Henry (2022). After six weeks, all non-inoculated control plants remained healthy while plants of both cultivars inoculated with the five isolates were severely wilted. Petiole assays yielded colonies identical in appearance to the inoculated isolates. For Fof race 1-inoculated plants, wilt symptoms were observed in Monterey but not in Fronteras. This experiment was repeated with P2, P3, P12, and P13 on another FW1 cultivar, San Andreas, and the same results were observed. To our knowledge, this is the first report of F. oxysporum f. sp. fragariae race 2 in California. Losses to Fusarium wilt are likely to increase until genetic resistance to this strain of Fof race 2 is deployed in commercially viable cultivars.

Ganoderma zonatum causes butt rot of areca (Dypsis lutescens) and robellini (Phoenix roebelenii) palms in Florida.

Ganoderma butt rot is a lethal disease of palms (Arecaceae) prevalent in palm-growing regions in the US that infects at least 58 species of palms (Elliott and Broschat 2001). Early symptoms appear as wilting of older fronds in the lower part of the canopy, and as disease progresses, wilting advances to younger leaves higher in the canopy towards the unopened spear leaf eventually killing the palm. A characteristic sign of the disease is the appearance of fruiting bodies (basidiomata) at the base of the palm trunk close to soil line. Ganoderma butt rot disease was detected on clustering palm species, areca palms, with 9 (82%) clusters showing Ganoderma basidiocarps and dead stumps, and mortality was observed in 5 (45%) clusters. A sterile scalpel was used to transfer the context tissue from Ganoderma basidiomata to full-strength potato dextrose agar selective media supplemented with streptomycin (100 mg/l), lactic acid (2 ml/l) and benomyl (4 mg/l). The pure culture for isolate GAN-33 was grown at 28°C in complete darkness for 10 days. The fungal colony was ivory white in color that grew radially as a dense mycelial mat without any sporulation. To establish the identity of the fungus, DNA was extracted using the Qiagen DNeasy PowerSoil kit (Cat. #12888). Three barcoding genes, nuclear ribosomal DNA internal transcribed spacer (ITS) region, RNA polymerase II subunit 2 (rpb2) and translation elongation factor 1α (tef1α) were amplified using primers ITS1/ITS4 (White et al 1990), bRPB2-6f/bRPB2-b7.1R (Matheny et al 2007) and EF1-983F/EF1-2212R (Matheny et al 2007), respectively. The sequences were deposited in GenBank, accession numbers KX853442, KX853466 and KX853491 for ITS, rpb2 and tef1α, respectively (Elliott et al 2018). Comparison to the NCBI nucleotide sequence database identified isolate GAN-33 as Ganoderma zonatum based on 100, 99 and 99% similarity to ITS, rpb2 and tef1α sequences, respectively. Pathogenicity of G. zonatum isolate GAN-33 was determined on 1-year old seedlings of areca palm (Dypsis lutescens) and pygmy date palm (Phoenix roebelenii). Ganoderma zonatum inoculum was prepared by transferring two-week old cultures to autoclaved wheat berries and allowed to colonize for two weeks. Seedlings were gently removed from the pot and the roots were trimmed before placing them back in the pot ensuring that the roots were in contact with the G. zonatum colonized wheat berries. The inoculated and control seedlings were maintained in a growth chamber at 28°C 60% RH (daytime) and 24°C 50% RH (night time), 12h:8h light:dark period, and watered twice a week. Initial wilting symptoms started appearing approximately one month after inoculation and mortality was observed for a total of four seedlings at three months after inoculation i.e., 2 out of 3 G. zonatum inoculated seedlings died for both areca and robellini palms, whereas the non-inoculated areca and robellini palm control seedlings remained healthy and alive. The pathogen was re-isolated from inoculated roots, and its identity was confirmed by colony morphology and PCR using G. zonatum specific primers (Chakrabarti et al 2022). To the best of our knowledge this is the first report establishing G. zonatum as the pathogen responsible for Ganoderma butt rot of palms.

Efficacy of Non-Conventional Chemical Inducers against Fusarium oxysporum Inciting Wilt in Chilli

Chilli wilt incited by Fusarium oxysporum is the most devastating disease, causing substantial yield losses throughout chilli growing areas of the world. Fusarium oxysporum is a soil-borne fungus which survives for several years in soil. The disease is favoured by high temperature and high moisture, this condition plays significant role in development of wilt symptoms. The disease management using chemicals is economically not feasible and is also unsafe for the environment. Due to growing public awareness to environment and human health, need of the hour is to find alternate approaches for disease management. Therefore, to investigate induction of systemic resistance against Fusarium oxysporum in chilli. Different non-conventional chemical inducers like salicylic acid (SA), jasmonic acid (JA), Indole acetic acid (IAA), Indole butyric acid (IBA), chitosan, salicylic acid nano formulation and chitosan nano formulation were used in an experiment conducted in Department of plant pathology, Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana. These non-conventional chemicals with four concentrations 25, 50, 75 and 100 µg/ml were evaluated for their antifungal activity against Fusarium oxysporum under in vitro condition using poison food technique with four replications. It was found that the highest mycelial growth inhibition was observed in salicylic acid nano formulation (79.73%) at 100 µg/ml concentration followed by Jasmonic acid (77.12%) at 100 µg/ml concentration and the lowest growth inhibition was recorded in Indole acetic acid (27.15%) at 100 µg/ml concentration. The same trend was observed when chilli varieties Pusa Jwala (susceptible) and Pusa Sadabahar (moderately resistant) were tested with the two best and the least effective non-conventional chemical inducer obtained in vitro for measuring the disease severity in screen house. This insight clarifies the scope of using non-conventional chemical inducers as an effective disease management strategy against Fusarium wilt of chilli.