Molecular characterization by PCR-ITS technique of Fusarium oxysporum isolated from tomato in Baghdad city

The present study aimed to estimate the pathogenesis of Fusarium oxysporum isolates and identify them by molecular technique Polymerase Chain Reaction-Internal Transcript Spacer (PCR-ITS). A total of thirty Fusarium oxysporum isolates were isolated from infected tomato plants and diagnosed depending on 'morphological characteristics. The pathogenicity test was performed for thirty Fusarium oxysporum isolates. So it was procedure steps that Genomic DNA was extracted from seven F. oxysporum isolates according to pathogenicity test by using'' ZR Fungal/Bacterial DNA'' MiniPrep™ kit. Seven F. oxysporum isolates showed high pathogenicity. The concentration and purity of the DNA extracted from the seven F. oxysporum isolates was (150 – 201) ng/µl and (1.4 -1.9) respectively. ITS gene was amplified by using PCR-ITS The sequencing results of amplified product of ITS gene from Fusarium oxysporum isolates indicated that all the seventh sequenced isolates where from one Formae speciales of Fusarium oxysporum as Fusarium oxysporum f. sp. Lycopersici. These results have been devoted to study genetic variability among the species involved in study. Molecular markers are highly important'' to focus on F. oxysporum isolates below'' the species level therefore, the DNA array containing "genus, species'' and forma specialis specific" detector'' for the detection and identification ''of F. oxysporum. Also, Cluster analysis and phylogenetic tree depending on genetic distance revealed the genetic relationship between the thirteen F. oxysporum isolates and confirmed sequencing analysis. [email protected]

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Morpho-molecular identification and pathogenicity test on fungal parasites of guava root-knot nematode eggs in Lampung, Indonesia

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d210334 ◽

2020 ◽

Vol 21 (3) ◽

Author(s):

I Gede Swibawa I Gede ◽

YUYUN FITRIANA ◽

SOLIKHIN ◽

RADIX SUHARJO ◽

F.X. SUSILO ◽

...

Keyword(s):

Molecular Identification ◽

Morphological Characteristics ◽

Pathogenicity Test ◽

Root Knot Nematode ◽

Commercial Products ◽

Tomato Plants ◽

Greenhouse Test ◽

Nematode Eggs ◽

Fungal Parasites

Abstract. Swibawa IG, Fitriana Y, Solikhin, Suharjo R, Susilo FX, Rani E, Haryani MS, Wardana RA. 2020. Morpho-molecular identification and pathogenicity test on fungal parasites of guava root-knot nematode eggs in Lampung, Indonesia. Biodiversitas 21: 1108-1115. This study aimed to obtain and discover the identity of the species of fungal egg parasites of root-knot nematodes (RKN), which have a high pathogenic ability causing major losses in vegetable crops. The exploration of the fungi was carried out in 2016 and 2018 from Crystal guava plantations in East Lampung, Central Lampung, Tanggamus, and NirAma, a commercial product that has been used for controlling Meloidogyne sp. in Indonesia. Identification was carried out based on morphological characteristics and molecular-based gene sequential analysis of Intergenic Transcribed Spacer (ITS) 1 and ITS 4. A pathogenicity test was carried out in vitro and in a greenhouse using tomato plants as indicator plants. In the in vitro test, observations were made on the percentage of infected RKN eggs. The observations in the greenhouse test were carried out on RKN populations in the soil and roots of tomato plants, root damage (root knots), and damage intensity due to RKN infection. The exploration resulted in five isolates of fungal egg parasites of RKN from the guava plantations in East Lampung (2), Central Lampung (1), Tanggamus (1), and from the isolation results of commercial products (1). The isolates were given codes as B4120X (PT GGP PG1), B3010 (PT GGP PG4), B412G (PT GGP PG 4), B01TG (Tanggamus), and BioP (Commercial products). Based on their morphological characteristics, the isolates were classified into the genus of Paecilomyces. The results of molecular identification showed that the discovered fungi were Purpureocillium lilacinum (Thom.) Luangsa Ard. (Syn. Paecilomyces lilacinus (Thom.) Samson.). Based on the in vitro tests, the five fungal isolates were able to parasitize RKN eggs at 86.4-100%. In the greenhouse test, all isolates significantly suppressed nematode populations in the soil and tomato roots, inhibited the formation of root knots, and produced lower damage intensity compared to controls. Among all the isolates tested, B01TG had the best ability to infect nematode eggs (99.5%), suppressing the formation of root knots, nematode population in the soil and the roots of tomato plants, and the damage intensity compared to other isolates.

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Current Classification and Diversity of Fusarium Species Complex, the Causal Pathogen of Fusarium Wilt Disease of Banana in Malaysia

Agronomy ◽

10.3390/agronomy11101955 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1955

Author(s):

Anysia Hedy Ujat ◽

Ganesan Vadamalai ◽

Yukako Hattori ◽

Chiharu Nakashima ◽

Clement Kiing Fook Wong ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Fusarium Wilt ◽

Species Complex ◽

Phylogenetic Trees ◽

Fusarium Species ◽

Morphological Characteristics ◽

Pathogenicity Test ◽

Fusarium Spp ◽

Specific Primers ◽

Fusarium Oxysporum Species Complex

The re-emergence of the Fusarium wilt caused by Fusarium odoratissimum (F. odoratissimum) causes global banana production loss. Thirty-eight isolates of Fusarium species (Fusarium spp.) were examined for morphological characteristics on different media, showing the typical Fusarium spp. The phylogenetic trees of Fusarium isolates were generated using the sequences of histone gene (H3) and translation elongation factor gene (TEF-1α). Specific primers were used to confirm the presence of F. odoratissimum. The phylogenetic trees showed the rich diversity of the genus Fusarium related to Fusarium wilt, which consists of F. odoratissimum, Fusarium grosmichelii, Fusarium sacchari, and an unknown species of the Fusarium oxysporum species complex. By using Foc-TR4 specific primers, 27 isolates were confirmed as F. odoratissimum. A pathogenicity test was conducted for 30 days on five different local cultivars including, Musa acuminata (AAA, AA) and Musa paradisiaca (AAB, ABB). Although foliar symptoms showed different severity of those disease progression, vascular symptoms of the inoculated plantlet showed that infection was uniformly severe. Therefore, it can be concluded that the Fusarium oxysporum species complex related to Fusarium wilt of banana in Malaysia is rich in diversity, and F. odoratissimum has pathogenicity to local banana cultivars in Malaysia regardless of the genotype of the banana plants.

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First Report of Fusarium Wilt on Philotheca myoporoides Caused by Fusarium oxysporum in Italy

Plant Disease ◽

10.1094/pdis-03-11-0243 ◽

2011 ◽

Vol 95 (7) ◽

pp. 877-877 ◽

Cited By ~ 1

Author(s):

G. Polizzi ◽

D. Aiello ◽

V. Guarnaccia ◽

A. Vitale ◽

G. Perrone ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Fusarium Wilt ◽

Morphological Characteristics ◽

Plant Production ◽

Pathogenicity Test ◽

Gene Sequences ◽

Pcr Assay ◽

First Report ◽

Gene Coding ◽

Eastern Australia

Philotheca myoporoides (DC.) M.J. Bayly (previously known as Eriostemon myoporoides), commonly called long-leaf waxflower and native to eastern Australia (Rutaceae family), is a hardy compact shrub or small tree occurring in subtropical to cool temperate regions. P. myoporoides is cultivated in Sicily (Italy) for its ornamental appeal. During April of 2010, a widespread wilting was observed on approximately 80% of 2,000 1-year-old, potted long-leaf waxflower plants grown in a commercial nursery near Catania (eastern Sicily, Italy). Internally, symptomatic plants had conspicuous vascular brown discoloration from the crown to the canopy. Diseased crown and stem tissues of 20 plants were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissues. Colonies with white or light purple aerial mycelia and violet pigmentation on the underside of the cultures developed after 9 days. On carnation leaf agar, 20 single-spore isolates produced microconidia on short monophialides, macroconidia that were three to five septate with a pedicellate base, and solitary and double-celled or aggregate chlamydospores. A PCR assay was conducted on one representative isolate (ITEM 13490) by analyzing sequences of the benA gene (coding β-tubulin protein) and CaM gene (coding calmodulin protein) using the primers reported by O'Donnell et al. (1). The benA gene sequences of ITEM 13490 (GenBank No. FR828825) exhibited an identity of 100% to Fusarium oxysporum f. sp. radicis-lycopersici strain ATCC 52429 (GenBank No. DQ092480). CaM gene sequences of ITEM 13490 (GenBank No. FR828826) exhibited an identity of 99.6% to F. oxysporum strain ITEM 2367 (GenBank No. AJ560774). Morphological characteristics of the 20 isolates, as well as the PCR assay on a representative strain, identified the isolates associated with disease symptoms as F. oxysporum Schlechtend.:Fr. A pathogenicity test was performed by placing two 1-cm2 plugs of PDA from 9-day-old mycelial cultures near the crown on potted, healthy, 2-month-old cuttings of P. myoporoides. Thirty plants were inoculated with strain ITEM 13490 and the same number of plants served as noninoculated controls. All plants were enclosed for 4 days in plastic bags and placed in a growth chamber at 25 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 23 to 27°C. First symptoms, which were identical to those observed in the nursery, developed on one plant 15 days after inoculation. Wilting was detected on all plants after 30 days. Control plants remained symptomless. F. oxysporum was successfully reisolated from symptomatic crown and stem tissues and identified as described above, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum causing disease of P. myoporoides worldwide. Moreover, this pathogen was recently reported in the same nursery on Eremophila sp. (2), confirming the presence of Fusarium wilt as a potential threat to ornamental plant production in this area, and necessitates the innovation and development of disinfection methods for alveolar trays, greenhouses, and various propagation materials to reduce future disease outbreaks. References: (1) K. O'Donnell et al. Mycoscience 41:61, 2000. (2) G. Polizzi et al. Plant Dis 94:1509, 2010.

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First Report of Fusarium Wilt in Blueberry (Vaccinium corymbosum) Caused by Fusarium oxysporum in China

Plant Disease ◽

10.1094/pdis-02-14-0167-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1158-1158 ◽

Cited By ~ 1

Author(s):

Y. H. Liu ◽

T. Lin ◽

C. S. Ye ◽

C. Q. Zhang

Keyword(s):

Fusarium Oxysporum ◽

Infected Plant ◽

Morphological Characteristics ◽

Vaccinium Corymbosum ◽

Morphological Characterization ◽

Internal Transcribed Spacers ◽

Fluorescent Light ◽

Pathogenicity Test ◽

Conidial Suspension ◽

First Report

Blueberry (Vaccinium corymbosum) production is developing quickly in China with about 20,000 ha presently cultivated. In 2010 in Lin'an, Zhejiang Province, plants developed an apparently new disease of blueberry (cv. Duke) with symptoms consisting of wilting of foliage, stunting of plants, and reduced fruit yields. Internal vascular and cortical tissues of plant crowns showed a brown to orange discoloration. Approximately 3% of the plants in the commercial plantings were affected and eventually died after 50 to 60 days. Infected plant samples (stems and roots) collected from different fields were surface sterilized with 1.5% sodium hypochlorite for 2 min, rinsed in water, plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 1 week. Single conidium cultures were consistently isolated and cultured on acidified PDA (APDA) for morphological characterization (1,2). Colonies were light with purple mycelia, and beige or orange reverse colony colors developed after 7 days incubation at 25°C. Colonies producing abundant microconidia and macroconidia. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.9 to 9.6 × 1.1 to 3.4 μm). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (28.6 to 37.5 × 3.3 to 4.2 μm). Morphology and development of macroconidia and microconida were consistent with a description of Fusarium oxysporum Schltdl (1,2). The ribosomal internal transcribed spacers ITS1 and ITS2 of eight isolates were amplified using primers ITS1/ITS4 on DNA extracted from mycelium and nucleotide sequences showed 100% similarity to that of F. oxysporum. To confirm pathogenicity, 20 blueberry plants (cv. Duke) were inoculated by dipping the roots into a conidial suspension (107 conidia per ml) for 30 min. The inoculated plants were transplanted into pots containing sterilized peat and maintained at 25°C and 100% relative humidity in a growth chamber with a daily 12-h photoperiod of fluorescent light. The pathogenicity test was conducted twice. Within 40 days, all inoculated plants developed wilt symptoms similar to that observed in the field. No symptoms were observed on plants dipped into distilled water. The fungus was successfully re-isolated from crowns and roots cultured on APDA, exhibiting morphological characteristics identical to F. oxysporum (1,2), confirming Koch's postulates. To our knowledge, this is the first report of blueberry wilt caused by Fusarium. References: (1) P. M. Kirk et al. The Dictionary of the Fungi, 10th edition, page 159. CABI Bioscience, Wallingford, UK, 2008. (2) W. C. Snyder and H. N. Hansen. Am. J. Bot. 27:64, 1940.

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Morphological aspects and effect of carbon sources in the physiology of Fusarium oxysporum f. sp. passiflorae

Emirates Journal of Food and Agriculture ◽

10.9755/ejfa.2018.v30.i1.1588 ◽

2018 ◽

pp. 77 ◽

Cited By ~ 1

Author(s):

Aricleia de Moraes Catarino ◽

Antonia A. C. Rodrigues, Machado Gois de Oliveira ◽

Diogo H.S. Sardinha Gomes da Costa

Keyword(s):

Fusarium Oxysporum ◽

Mycelial Growth ◽

Culture Medium ◽

Carbon Sources ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Morphological Characters ◽

Pathogenicity Test ◽

Morphological Aspects ◽

Yellow Passion Fruit

The objective of this study was to evaluate the morphological and physiological characteristics of F. oxysporum f. sp. passiflorae. Isolates were obtained from yellow passion fruit plants with fusariosis symptoms grown in four production poles. The pathogenicity test was carried out on seedlings of this crop, under greenhouse conditions. The morphological characters evaluated were the size of macroconidia and microconidia in single conidiophores or in false heads; number of septa, presence of basal cell in the macroconidia and chlamydospores, and the aspects of the colonies of the isolates cultivated in PDA culture medium for aerial mycelium formation and colony coloration. The carbon sources: starch, lactose, maltose and sucrose were tested for the three most virulent isolates obtained in the pathogenicity test. The mycelial growth was obtained through readings every 24 hours of the diameter of the colony in two diametrically opposite directions. Sporulation determination was performed by counting spores in a Neubauer chamber. The isolate Fusarium oxysporum f. sp. passiflorae 4 (FOP 4) was the most virulent. There was variation in the morphological characteristics of all the isolates. The starch provided the highest mycelial growth and the isolates showed preferences for a particular carbon source in the sporulation test.

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Detection of tomatinase from Fusarium oxysporum f. sp. lycopersici in infected tomato plants

Phytochemistry ◽

10.1016/s0031-9422(97)00170-2 ◽

1997 ◽

Vol 45 (7) ◽

pp. 1371-1376 ◽

Cited By ~ 9

Author(s):

Khalid Lairini ◽

Manuel Ruiz-Rubio

Keyword(s):

Fusarium Oxysporum ◽

Tomato Plants ◽

Infected Tomato

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First Report of Dry and Soft Rot of Cereus marginatus var. cristata Caused by Fusarium oxysporum in Italy

Plant Disease ◽

10.1094/pdis-06-14-0574-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1441-1441 ◽

Cited By ~ 2

Author(s):

A. Garibaldi ◽

P. Pensa ◽

D. Bertetti ◽

G. Ortu ◽

M. L. Gullino

Keyword(s):

Relative Humidity ◽

Fusarium Oxysporum ◽

Apical Cell ◽

Morphological Characteristics ◽

Soft Rot ◽

The United States ◽

Selective Medium ◽

Economic Losses ◽

Pathogenicity Test ◽

Dry Rot

During the winter of 2013, 50% of 20,000 plants of Cereus marginatus var. cristata, Cactaceae family, grown in a commercial farm located in Liguria (northern Italy) showed symptoms of a dry or soft rot. In the case of dry rot, affected plants showed on the stem superficial necrosis and dry rot, irregularly shaped, 1 to 10 mm, while epidermal and cortical tissues were wounded. Affected plants survived but they lost ornamental value. In the case of soft rot, associated with conditions of higher relative humidity, rots on the stem extended as far as 4 cm in width. The internal part of bark, cambium, and xylem tissues as far as about 3 cm in depth was rotted. Vascular tissues were not discolored. Plants died in about 20 days. A Fusarium sp. was consistently isolated from symptomatic tissue on Komada selective medium (2) from plants showing soft rot. The isolates were purified and subcultured on potato dextrose agar (PDA). On PDA, the cultures produced a thick and soft growth of white to light pink mycelium and pale pink pigments in the agar. On Spezieller Nährstoffarmer agar (SNA), cultures produced short monophialides with unicellular, ovoid-elliptical microconidia measuring 3.7 to 8.2 × 1.7 to 3.5 (average 5.4 × 2.5) μm. On carnation leaf-piece agar (CLA), chlamydospores were abundant, terminal or intercalary, single or paired, but frequently also aggregated. On the same medium, at temperatures ranging from 20 to 24°C (14 h daylight, 10 h dark), cultures produced light orange sporodochia with macroconidia. These were 3 to 4 (sometimes 5) septate, nearly straight with a foot-shaped basal cell and a short apical cell, and measured 28.5 to 41.4 × 3.3 to 4.9 (average 35.0 × 4.0) μm. Such characteristics are typical of Fusarium oxysporum Schlechtendahl emend. Snyder & Hansen (3). Amplification of the internal transcribed spacer (ITS) of the rDNA using primers ITS1/ITS4 yielded a 504-bp amplicon (GenBank Accession No. KJ909935). Sequencing and BLASTn analysis of this amplicon showed a 100% homology with the sequence of F. oxysporum KC304802. To confirm pathogenicity, two Fusarium isolates were tested. For each isolate, three 2-year-old healthy plants of C. marginatus were inoculated by introducing into lesions (4 lesions/plant) artificially produced on the stem sterile needles contaminated with the pathogen (4). Inoculum was obtained from pure cultures grown on PDA. Control plants were punctured with sterile needles without inoculum. All the plants were placed in a greenhouse, at temperatures ranging between 16 and 24°C. For both tested strains, the first necrosis of stem tissues developed around the needles 7 days after the artificial inoculation, while non-inoculated plants remained healthy. Then, necrosis extended causing soft rot on plants maintained at relative humidity ranging from 55 to 65%. F. oxysporum identified by morphological characteristics was consistently isolated from symptomatic plants. The pathogenicity test was conducted twice. F. oxysporum has been reported on Cereus sp. in the United States and on C. peruvianus monstruosus in Italy (1). Currently, this disease is present in a few commercial nurseries in Liguria, although it could spread further and cause important economic losses. References: (1) A. Garibaldi et al. Plant Dis. 95:877, 2011. (2) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (4) V. Talgø and A. Stensvand. OEPP/EPPO Bulletin 43:276, 2013.

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