First report of damping off disease caused by Fusarium oxysporum on Pinus pinea in Jordan

Forests of Jordan are located in the northern and southern parts of the country with 60% and 40%, respectively. Natural forests constitute about 75% in the northern part and 25% in the southern part. There are many types of forest trees in Jordan including pines (Pinus spp.), juniper (Juniperus), cypress (Cupressus), oak (Querus), acacia (Acacia), and Christ's thorn jujube (Ziziphus). There are three species of Pinus: P. halopensis (native), P. pinea (introduced), and P. canariensis (introduced) (Ministry of Agriculture, 2013). P. pinea is considered one of the most important components of Jordan's natural forests. Due to its adaptability, lack of environmental requirements and ease of cultivation, its cultivation has been expanded in all parts of Jordan. P. pinea cultivation prevent soil erosion and combating desertification. P. pinea seeds are used in making sweets and many popular foods. In the end of 2019, wilting and damping-off symptoms were noticed in 50 % of P. pinea seedlings nurseries (personal communication, November 2019). Six-month old P. pinea seedlings with visible symptoms of damping-off were collected between May and July 2020 from a pine nursery located in Amman Province, Jordan (32° 0′ 40.4316″ N, 135° 52′ 20.3628″ E). Thirty-two seedlings with different severities of the disease were selected for the isolation of root pathogens. Two root samples from each seedling were surface-sterilized using 1% sodium hypochlorite for 3 - 5 minutes and then rinsed with sterile distilled water. Root samples were subsequently cut into small pieces (1- to 2 cm long sections) and then placed on potato dextrose agar (PDA) supplemented with 5 mg/L streptomycin sulphate. Petri dishes were incubated in a growth chamber at 25±2°C for seven days and sub-cultured by hyphal tipping. The cultural and conidial morphology of 7-day old mycelia were observed for the isolates using an optical microscope (LEICA, ICC50 HD, Switzerland). For morphological identification of Fusarium, 200 measurements of microconidia, macroconidia and chlamydospores were conducted. The Fusarium isolates had a color of purple-violet mycelium growth in a PDA culture medium (Figure 1 A and B). Macroconidia had 3-5 septa with a foot- shaped basal cell. They were sickle-shaped, hyaline, and thin-walled with dimensions of 20-42 x 3.2-5.5 μm (Figure 1C). Microconidia were short, elliptic to oval unicellular, and with sharp unbranched monophyalides with an average dimension of 5.0–17.0×2.3–5.1 μm (Figure 1D, 1E). Older mycelia developed a large number of terminal chlamydospores (7.2 to 14.1 µm) that were intercalary and rough-walled (Figure 1F). All the characteristics agreed with those recorded by Leslie et al. (2006) and Nelson et al. (1983) for Fusarium oxysporum. Representative isolate (FoxypineJO2020-01) was selected for molecular identification. The DNA was extracted, amplified using the translation elongation factor 1-α (EF1α) gene (EF1/EF2) (O’Donnell et al., 1998), and sequenced at Macrogen Inc, South Korea. Forward and reverse sequences were received, assembled and consensus sequence was produced using BioEdit sequence alignment editor (Hall, 1999). The consensus sequence was BLASTn on the NCBI website (https://www.ncbi.nlm.nih.gov) and was 100% similar with F. oxysporum accession KC622308.1. Phylogenetic analysis was conducted using MEGA 7.0.26 (Kumar et al. 2016) with 1000 bootstrap values and correlated the representative isolate with the accession KC622308.1 (Figure 2). The isolated sequence was deposited in the GenBank and accession number was assigned (MW057934). Koch's postulates were fulfilled using FoxypineJO2020-01 isolate to confirm the Fusarium oxysporum as the causal agent of Pinus pinea damping-off. One-month-old seedlings of P. pinea were soaked in spore suspension of 1 × 106 spores/ml for 10 minutes. Seedlings were placed in 25cm x 20cm deep plastic pots filled with a sterile mixture of peat moss, perlite, and vermiculite (60:20:20). Controlled by thirty seedlings of P. pinea soaked in distilled water. Planted seedlings were incubated at 25 ± 2°C with a 12: 12 hrs light/dark period. Seedlings of P. pinea inoculated with spores gradually showed symptoms similar to those of naturally diseased infected plants (Figure 3, 4). The inoculated pathogen was successfully re-isolated from roots of the diseased seedlings. The uppermost leaves began to wilt (Figure 4c), and the roots had darkened at 25 days after inoculation (Figure 4d). By 40 days after inoculation, the entire seelings were discolored and dead (Figure 4e). Furthermore, the roots became dark and peeled (Figure 4f). These symptoms matched those described by (Machón et al., 2009) and (Luo and Yu 2020). Control P. pinea seedlings remained asymptomatic (Figure 4a, b). To our knowledge, this is the first report of F. oxysporum on P. pinea in Jordan. No previous disease notes were reported on P. pinea seedlings in Jordan. The pathogen can cause significant economic losses to P. pinea as well as to other types of Pinus spp. whether in nurseries or forests in Jordan. Therefore, for disease control in nurseries, it is extremely important to determine the onset time, decrease the incidence (Gordon et al. 2015) and identify the infection source (Morales-Rodriguezv et al. 2018). Future surveys need to be conducted on forest trees in selected forest and biosphere reserves that show tree decline to identify major forest fungal pathogens in Jordanian forests.

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First Report of Diaporthe hongkongensis Causing Fruit rot on Peach (Prunus persica) in China

Plant Disease ◽

10.1094/pdis-07-20-1505-pdn ◽

2021 ◽

Author(s):

Zhou Zhang ◽

Zheng Bing Zhang ◽

Yuan Tai Huang ◽

FeiXiang Wang ◽

Wei Hua Hu ◽

...

Keyword(s):

Prunus Persica ◽

Fruit Rot ◽

Hunan Province ◽

Post Inoculation ◽

Distilled Water ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Representative Isolate ◽

Rot Disease

Peach [Prunus persica (L.) Batsch] is an important deciduous fruit tree in the family Rosaceae and is a widely grown fruit in China (Verde et al., 2013). In July and August 2018, a fruit rot disease was observed in a few peach orchards in Zhuzhou city, the Hunan Province of China. Approximately 30% of the fruit in more than 400 trees was affected. Symptoms displayed were brown necrotic spots that expanded, coalesced, and lead to fruit being rotten. Symptomatic tissues excised from the margins of lesions were surface sterilized in 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile distilled water three times, and incubated on potato dextrose agar (PDA) at 26°C in the dark. Fungal colonies with similar morphology developed, and eight fungal colonies were isolated for further identification. Colonies grown on PDA were grayish-white with white aerial mycelium. After an incubation period of approximately 3 weeks, pycnidia developed and produced α-conidia and β-conidia. The α-conidia were one-celled, hyaline, fusiform, and ranged in size from 6.0 to 8.4 × 2.1 to 3.1 μm, whereas the β-conidia were filiform, hamate, and 15.0 to 27.0 × 0.8 to 1.6 μm. For molecular identification, total genomic DNA was extracted from the mycelium of a representative isolate HT-1 and the internal transcribed spacer region (ITS), β-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), calmodulin (CAL), and histone H3 gene (HIS) were amplified and sequenced (Meng et al. 2018). The ITS, TUB, TEF1, CAL and HIS sequences (GenBank accession nos. MT740484, MT749776, MT749778, MT749777, and MT749779, respectively) were obtained and in analysis by BLAST against sequences in NCBI GenBank, showed 99.37 to 100% identity with D. hongkongensis or D. lithocarpus (the synonym of D. hongkongensis) (Gao et al., 2016) (GenBank accession nos. MG832540.1 for ITS, LT601561.1 for TUB, KJ490551.1 for HIS, KY433566.1 for TEF1, and MK442962.1 for CAL). Pathogenicity tests were performed on peach fruits by inoculation of mycelial plugs and conidial suspensions. In one set, 0.5 mm diameter mycelial discs, which were obtained from an actively growing representative isolate of the fungus on PDA, were placed individually on the surface of each fruit. Sterile agar plugs were used as controls. In another set, each of the fruits was inoculated by application of 1 ml conidial suspension (105 conidia/ml) by a spray bottle. Control assays were carried out with sterile distilled water. All treatments were maintained in humid chambers at 26°C with a 12-h photoperiod. The inoculation tests were conducted twice, with each one having three fruits as replications. Six days post-inoculation, symptoms of fruit rot were observed on inoculated fruits, whereas no symptoms developed on fruits treated with agar plugs and sterile water. The fungus was re-isolated and identified to be D. hongkongensis by morphological and molecular methods, thus fulfilling Koch’s Postulates. This fungus has been reported to cause fruit rot on kiwifruit (Li et al. 2016) and is also known to cause peach tree dieback in China (Dissanayake et al. 2017). However, to our knowledge, this is the first report of D. hongkongensis causing peach fruit rot disease in China. The identification of the pathogen will provide important information for growers to manage this disease.

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First Report of Fusarium Wilt of Canary Island Date Palm Caused by Fusarium oxysporum f. sp. canariensis in Louisiana

Plant Disease ◽

10.1094/pdis-03-11-0239 ◽

2011 ◽

Vol 95 (9) ◽

pp. 1192-1192 ◽

Cited By ~ 3

Author(s):

R. Singh ◽

A. Castro ◽

D. M. Ferrin ◽

R. S. Harris ◽

B. Olson

Keyword(s):

South Carolina ◽

Fusarium Oxysporum ◽

New Orleans ◽

Fusarium Wilt ◽

Canary Island ◽

Date Palm ◽

Distilled Water ◽

Date Palms ◽

First Report ◽

Vascular Discoloration

Canary Island date palm (Phoenix canariensis Hort. Ex Chabaud) is a signature palm planted in New Orleans, LA. Currently, the city has approximately 1,000 mature Canary Island date palms. During the fall of 2009, 153 palms were inspected with 27 palms exhibiting typical symptoms of Fusarium wilt. Symptoms included one-sided death and a reddish brown streak on the rachis of affected fronds and death of the leaflets. Longitudinal sections of affected fronds showed vascular discoloration. Severely infected palms were completely dead. Small pieces of diseased tissue from five palms were surface sterilized with sodium hypochlorite (0.6%) for 2 to 3 min, then rinsed in sterile distilled water, blotted dry, and plated on potato dextrose agar (PDA). Fungal colonies on PDA produced a purple pigment, and both macro- and microconidia that are typical of Fusarium oxysporum were observed under a light microscope. A single-spore culture of isolate PDC-4701 was obtained. DNA from this isolate was extracted with a DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) and primers ef1 and ef2 were used to amplify and sequence the translation elongation factor 1-α gene (2). NCBI BLAST analysis of the 616-bp sequence resulted in 100% identity with F. oxysporum f. sp. canariensis isolates PLM-385B from Texas and PLM-511A from South Carolina (GenBank Accession Nos. HM 591538 and HM 591537, respectively). Isolate PDC-4701, grown on PDA for 2 weeks, was used to inoculate 10 9-month-old P. canariensis seedlings. An 18-gauge needle was used to inject 15 ml of a 107 conidia/ml suspension into the stem near the soil line. Each seedling was inoculated at two locations and covered with Parafilm at the inoculation sites. Ten control seedlings were injected with sterile distilled water in the same manner. Inoculated and control seedlings were maintained in a greenhouse at 28 ± 2°C. Leaves of all 10 inoculated seedlings started to wilt 3 months after inoculation. Internal vascular discoloration was observed and the pathogen was reisolated from the symptomatic seedlings. No symptoms developed on any of the 10 control seedlings. On the basis of morphology and DNA sequence data, this pathogen is identified as F. oxysporum f. sp. canariensis. Fusarium wilt of Canary Island date palm has been previously reported from California, Florida, Nevada, Texas, and South Carolina (1). To our knowledge, this is the first report of Fusarium wilt of Canary Island date palm caused by F. oxysporum f. sp. canariensis in Louisiana, extending its geographic range. The disease may adversely affect the tradition of planting Canary Island date palms in New Orleans. The sequence of isolate PDC-4701 has been submitted to the NCBI database (GenBank Accession No. JF826442) and a culture specimen has been deposited in the Fusarium Research Center culture collection (Accession No. O-2602) at the Pennsylvania State University, University Park, PA. References: (1) M. L. Elliott et al. Plant Dis. 95:356, 2011. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004.

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First Report on Ovate-leaf Atractylodes Damping-off Caused by Fusarium oxysporum species Complex in South Korea

Plant Disease ◽

10.1094/pdis-05-21-1107-pdn ◽

2021 ◽

Author(s):

Oliul Hassan ◽

Taehyun Chang

Keyword(s):

South Korea ◽

Fusarium Oxysporum ◽

Species Complex ◽

Disease Incidence ◽

Fusarium Species ◽

Morphological Characteristics ◽

Conidial Suspension ◽

Sterile Water ◽

Damping Off ◽

First Report

In South Korea, ovate-leaf atractylodes (OLA) (Atractylodes ovata) is cultivated for herbal medicine. During May to June 2019, a disease with damping off symptoms on OLA seedlings were observed at three farmer fields in Mungyeong, South Korea. Disease incidence was estimated as approximately 20% based on calculating the proportion of symptomatic seedlings in three randomly selected fields. Six randomly selected seedlings (two from each field) showing damping off symptoms were collected. Small pieces (1 cm2) were cut from infected roots, surface-sterilized (1 minute in 0.5% sodium hypochlorite), rinsed twice with sterile water, air-dried and then plated on potato dextrose agar (PDA, Difco, and Becton Dickinson). Hyphal tips were excised and transferred to fresh PDA. Six morphologically similar isolates were obtained from six samples. Seven-day-old colonies, incubated at 25 °C in the dark on PDA, were whitish with light purple mycelia on the upper side and white with light purple at the center on the reverse side. Macroconidia were 3–5 septate, curved, both ends were pointed, and were 19.8–36.62 × 3.3–4.7 µm (n= 30). Microconidia were cylindrical or ellipsoid and 5.5–11.6 × 2.5–3.8 µm (n=30). Chlamydospores were globose and 9.6 –16.3 × 9.4 – 15.0 µm (n=30). The morphological characteristics of present isolates were comparable with that of Fusarium species (Maryani et al. 2019). Genomic DNA was extracted from 4 days old cultures of each isolate of SRRM 4.2, SRRH3, and SRRH5, EF-1α and rpb2 region were amplified using EF792 + EF829, and RPB2-5f2 + RPB2-7cr primer sets, respectively (Carbone and Kohn, 1999; O'Donnell et al. 2010) and sequenced (GenBank accession number: LC569791- LC569793 and LC600806- LC600808). BLAST query against Fusarium loci sampled and multilocus sequence typing database revealed that 99–100% identity to corresponding sequences of the F. oxysporum species complex (strain NRRL 28395 and 26379). Maximum likelihood phylogenetic analysis with MEGA v. 6.0 using the concatenated sequencing data for EF-1α and rpb2 showed that the isolates belonged to F. oxysporum species complex. Each three healthy seedlings with similar sized (big flower sabju) were grown for 20 days in a plastic pot containing autoclaved peat soil was used for pathogenicity tests. Conidial suspensions (106 conidia mL−1) of 20 days old colonies per isolate (two isolates) were prepared in sterile water. Three pots per strain were inoculated either by pouring 50 ml of the conidial suspension or by the same quantity of sterile distilled water as control. After inoculation, all pots were incubated at 25 °C with a 16-hour light/8-hour dark cycle in a growth chamber. This experiment repeated twice. Inoculated seedlings were watered twice a week. Approximately 60% of the inoculated seedlings per strain wilted after 15 days of inoculation and control seedlings remained asymptomatic. Fusarium oxysporum was successfully isolated from infected seedling and identified based on morphology and EF-1α sequences data to confirm Koch’s postulates. Fusarium oxysporum is responsible for damping-off of many plant species, including larch, tomato, melon, bean, banana, cotton, chickpea, and Arabidopsis thaliana (Fourie et al. 2011; Hassan et al.2019). To the best of our knowledge, this is the first report on damping-off of ovate-leaf atractylodes caused by F. oxysporum in South Korea. This finding provides a basis for studying the epidemic and management of the disease.

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First Report of Mefenoxam Sensitivity and Pathogenicity of Phytophthora citricola Isolated from American Ginseng (Panax quinquefolium)

Plant Disease ◽

10.1094/pdis-92-12-1706a ◽

2008 ◽

Vol 92 (12) ◽

pp. 1706-1706

Author(s):

S. N. Hill ◽

O. P. Hurtado-Gonzales ◽

K. H. Lamour ◽

M. K. Hausbeck

Keyword(s):

Block Design ◽

Silica Sand ◽

Distilled Water ◽

Panax Quinquefolium ◽

Damping Off ◽

Tissue Samples ◽

First Report ◽

Zoospore Release ◽

Fluorescent Lighting

In March of 2004, stratified ginseng seeds from commercial Wisconsin gardens were planted in sterilized silica sand in a research greenhouse at Michigan State University. Following emergence, seedlings exhibiting wilting, damping off, and black stem lesions were observed. In the laboratory, symptomatic seedlings were rinsed with distilled water. Tissue samples were excised and embedded in water agar amended with ampicillin (100 mg/liter) and incubated at 25°C. In addition to the isolation of Phytophthora cactorum, a known pathogen of ginseng, P. citricola, (five isolates) also was identified from single-zoospore cultures based on morphology (2). One-week-old, dilute V8 agar cultures were used to obtain single zoospores. Cultures were flooded with 20 ml of sterilized distilled water chilled to 10°C and incubated at 25°C for 25 min to induce zoospore release. Zoospore suspensions were spread onto water agar plates, and after 24 h at 25°C, single germinating zoospores were selected at random and transferred to benomyl, ampicillin, rifampicin, and pentachloronitrobenzene (BARP)-amended V8 agar plates. Sequence analysis of the internal transcribed spacer (ITS) region 1 and 2 of the rDNA was also used to distinguish P. citricola from P. cactorum. A representative sequence for the isolates of P. citricola (NCBI Accession No. FJ217388) matched (100% similarity) a P. citricola isolate deposited in GenBank (Accession No. DQ486661). To screen P. citricola for in vitro response to mefenoxam, agar plugs (7-mm diameter) from 1-week-old V8 agar cultures incubated at 25°C under fluorescent lighting were placed in the center of each of two V8 agar plates amended with 0 and 100 ppm of mefenoxam (Ridomil Gold EC, 48% a.i., suspended in sterile distilled water and added to V8 agar cooled to 49°C). The plates were incubated at 25°C for 3 days under fluorescent lighting. Isolates were assigned a mefenoxam sensitivity rating based on the percentage of radial mycelial growth on the amended V8 agar when compared with the unamended control. Each of the five isolates was scored as mefenoxam resistant with growth on 100-ppm plates >30% of the controls. Koch's postulates were conducted for the isolates of P. citricola recovered from ginseng seedlings to confirm pathogenicity. Previously, P. citricola was reported as nonpathogenic to ginseng (1). Three-week-old, healthy ginseng seedlings were planted into 89- × 64-mm pots filled with autoclaved medium-particle vermiculite and maintained in the greenhouse under 63% shade cloth with temperatures between 18 and 26°C. Pots were arranged in a completely randomized block design with eight seedlings per isolate as replicates and watered as needed. A 2-ml inoculum suspension (approximately 104 zoospores) was injected into the potting medium at the stem base of each seedling. All of the isolates were pathogenic to ginseng seedlings with 60% of inoculated seedlings per isolate exhibiting wilting, damping off, and blackened stems within 3 weeks after inoculation. P. citricola was reisolated from all inoculated plants. To our knowledge, this is the first report of P. citricola pathogenic on ginseng. References: (1) T. W. Darmono et al. Plant Dis. 75:610, 1991. (2) D. C. Erwin and O. K. Ribeiro. Page 96 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN. 1996.

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First Report of Ginger Rhizome Rot Caused by Fusarium oxysporum in China

Plant Disease ◽

10.1094/pdis-07-13-0729-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 282-282 ◽

Cited By ~ 6

Author(s):

Y. Li ◽

L. D. Chi ◽

L. G. Mao ◽

D. D. Yan ◽

Z. F. Wu ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Casein Hydrolysate ◽

Zingiber Officinale ◽

Selective Medium ◽

Shandong Province ◽

Pathogenicity Test ◽

Distilled Water ◽

Dry Rot ◽

First Report ◽

Rhizome Rot

Ginger (Zingiber officinale Roscoe) is an important commercial crop that is planted in 60,000 to 70,000 ha every year in Shandong Province, China. In 2010, rotted rhizomes of cultivar Laiwu Big Ginger were reported on 20 ha in Anqiu, Shandong Province, and yield losses of up to 70% were reported. The aboveground symptoms were the water-conducting portion of symptomatic rhizomes was discolored brown and had a black dry rot of the cortex tissues (3). Thirty symptomatic rhizomes were sampled from six fields in six farms. Komada's method (1) was used to isolate the pathogen. Ten pieces from each rhizome were washed with sterile distilled water and plated on Komada selective medium at 25°C. White fungal colonies turned orchid after 7 days of incubation. Two types of asexual spores were associated with the colonies: microconidia and macroconidia. The microconidia were the most abundantly produced spores and were oval, elliptical or kidney shaped, and produced on aerial mycelia. Macroconidia had three to five cells and gradually pointed or curved edges, varied in size from 3 to 5 × 19 to 36 μm. The rDNA of the internal transcribed spacer regions 1 and 2 and the 5.8S gene in five isolates were amplified using primers ITS1 and ITS4, and the nucleotide sequence was the same as isolate no. 3, which was deposited in GenBank (Accession No. KC594035). A BLAST search showed 99% identity with the strain Z9 of Fusarium oxysporum (EF611088). Pathogenicity tests of five isolates were carried out in a greenhouse and the pathogenicity test of isolate no. 3 was selected for the method description. Ten 1-month-old ginger plants (cv. Laiwu Big Ginger) were grown in plastic pots (diameter 20 cm) with sandy soil and inoculated. Ten plants were used as untreated controls. Isolate no. 3 was grown on casein hydrolysate medium (4) for 72 h and the spores were harvested in sterile distilled water. Aqueous spore suspensions of isolate no. 3 were adjusted with deionized water to 1 × 108 CFU/ml as the inoculum. The prepared inoculum was injected with a syringe into the soil around the rhizome of ginger plants. Inoculated plants were placed in the greenhouse at 24 to 26°C and assessed for rhizome rot on the 14th day after inoculation. Disease severity was recorded based on a scale in which – = no symptoms; 1 = small lesions on seedlings, no rot; 2 = seedling rot; and 3 = plant dead. Similar rhizome rot symptoms were observed after inoculation. The inoculated isolate was re-isolated from diseased rhizomes, confirming its pathogenicity. To our knowledge, this is the first report of rhizome rot of ginger caused by F. oxysporum in China. Rhizome rot of ginger caused by Fusarium spp. is well known in Asian countries such as India (2). References: (1) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (2) V. Shanmugam et al. Biol Control. 66:1, 2013. (3) E. E. Trujillo. Diseases of Ginger (Zingiber officinale) in Hawaii, Circular 62, Hawaii Agricultural Experiment Station, University of Hawaii, December, 1964. (4) G. E. Wessman. Appl. Microbiol. 13:426, 1965.

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First Report of Fusarium wilt disease on Watermelon Caused by Fusarium oxysporum f. sp. niveum (FON) in Malaysia

Plant Disease ◽

10.1094/pdis-04-21-0780-pdn ◽

2021 ◽

Author(s):

Muhammad Ziaur Rahman ◽

Khairulmazmi Ahmad ◽

Yasmeen Siddiqui ◽

Norsazilawati Saad ◽

Tan Geok Hun ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Fusarium Wilt ◽

Cover Crops ◽

Fungal Pathogen ◽

Disease Incidence ◽

Wilt Disease ◽

Likelihood Method ◽

Distilled Water ◽

First Report ◽

Fusarium Wilt Disease

Fusarium wilt disease incited by Fusarium oxysporum f. sp. niveum (FON) is the utmost devastating soil-inhabiting fungal pathogen limiting watermelon (Citrullus lanatus) production in Malaysia and globally. The field disease survey of fusarium wilt was carried out during December 2019 and November 2020, in three major production areas (3 farmer fields per location) in Peninsular Malaysia namely, Mersing, Serdang and Kuantan and disease incidence of 30 and 45%, was recorded for each year, respectively. Infected watermelon plants showed symptoms such as vascular discoloration, brown necrotic lesions to the soil line or the crown, one-sided wilt of a plant, or a runner or the whole plant. Infected root and stem tissues, 1-2 cm pieces were surface sterilized with 0.6% NaOCl for 1 minute followed by double washing with sterile water. The disinfected tissues were air-dried and transferred onto semi-selective Komada’s medium (Komada 1975) and incubated for 5 days. The fungal colonies produced were placed on potato dextrose agar (PDA) to attain a pure culture and incubated at 25±2℃ for 15 days. The pure fungal colony was flat, round and light purple in color. Macroconidia were straight to slightly curved, 18.56-42.22 µm in length, 2.69-4.08 µm width, predominantly 3 septate and formed in sporodochia. Microconidia measured 6.16-10.86 µm in length and 2.49-3.83 µm in width, kidney-shaped, aseptate and were formed on short monophialides in false-heads. Chlamydospores were single or in pairs with smooth or rough walls, found both terminally or intercalary. To confirm their pathogenicity, two-week-old watermelon seedlings (cv. NEW BEAUTY) were dipped into spore suspension (1 ˟ 106 spores/ml) of representative isolates of JO20 (Mersing), UPM4 (Serdang) and KU41 (Kuantan) for 30 second and then moved into 10 cm diameter plastic pots containing 300 g sterilized soil mix. Disease symptoms were assessed weekly for one month. Control seedlings were immersed in sterile distilled water before transplanting. The inoculated seedlings showed typical Fusarium wilt symptoms like yellowing, stunted growth, and wilting, which is similar to the farmer field infected plants. However, the seedlings inoculated by sterile distilled water remained asymptomatic. The pathogen was successfully re-isolated from the infected seedlings onto Komada’s medium, fulfilling the Koch’s postulate. For the PCR amplification, primers EF-1 and EF-2 were used to amplify the tef1-α region. A Blastn analysis of the tef1-α sequences of the isolates JO20 (accession nos. MW315902), UPM4 (MW839560) and KU41 (MW839562) showed 100% similarity; with e-value of zero, to the reference sequences of F. oxysporum isolate FJAT-31690 (MN507110) and F. oxysporum f. sp. niveum isolate FON2 790-2 (MN057702). In Fusarium MLST database, isolates JO20, UPM4 and KU41 revealed 100% identity with the reference isolate of NRRL 22518 (accession no. FJ985265). Though isolate FJ985265 belongs to the f. sp. melonis, earlier findings had revealed Fusarium oxysporum f. sp. are naturally polyphyletic and making clusters with diverse groups of the Fusarium oxysporum species complex (O’Donnell et al. 2015). The isolates JO20, UPM4 and KU41 were identified as F. oxysporum f. sp. niveum based on the aligned sequences of tef1-α and molecular phylogenetic exploration by the maximum likelihood method. To the best of our knowledge, this is the first report of F. oxysporum f. sp. niveum as a causative pathogen of Fusarium wilt disease of watermelon in Malaysia. Malaysia enables to export watermelon all-year-round in different countries like Singapore, Hong-Kong, The United Arab Emirates (UAE), and Netherlands. The outburst of this destructive soil-borne fungal pathogen could cause hindrance to watermelon cultivation in Malaysia. Thus, growers need to choice multiple management tactics such as resistant varieties, cultural practices (soil amendments and solarization), grafting, cover crops and fungicide application to control this new pathogen.

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First Report of Seedling Stem Rot on Jinxianlian (Anoectochilus roxburghii) Caused by Fusarium oxysporum in China

Plant Disease ◽

10.1094/pdis-09-21-2066-pdn ◽

2021 ◽

Author(s):

Chuan-Qing Zhang ◽

X. Y. Chen ◽

Ya-hui Liu ◽

Dejiang Dai

Keyword(s):

Fusarium Oxysporum ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Morphological Features ◽

Stem Rot ◽

Broad Host Range ◽

Distilled Water ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Anoectochilus Roxburghii

Anoectochilus roxburghii is an important Chinese herbal medicine plant belonging to Orchidaceae and known as Jinxianlian. This orchid is cultivated and mostly adopted to treat diabetes and hepatitis. About 2 billion artificially cultivated seedlings of Jinxianlian are required each year and approximately $600 million in fresh A. roxburghii seedlings is produced in China. From 2011, sporadic occurrence of stem rot on Jinxianlian have been observed in greenhouses in Jinhua City (N29°05′, E119°38′), Zhejiang Province. In 2018, nearly 30% of seedlings of Jinxianlian grown in greenhouse conditions were affected by stem rot in Jinhua City. Symptoms initially occurred in the stem at the soil line causing dark discoloration lesions, rotted tissues, wilting, and eventually leading to the death of the plants. A total of 23 diseased seedlings collected from seven different greenhouses were surface sterilized with 1.5% sodium hypochlorite for 3 min, then rinsed in water. Pieces of tissues disinfected from each sample were plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 days (Kirk et al. 2008). A total of 19 isolates were recovered. They developed colonies with purple mycelia and beige or orange colors after 7 days of incubation under 25°C on PDA and carnation leaf agar (CLA) media (Kirk et al. 2008; Zhang et al. 2016). Colonies on PDA had an average radial growth rate of 3.1 to 4.0 mm /d at 25°C. Colony surface was pale vinaceous, floccose with abundant aerial mycelium. On CLA, aerial mycelium was sparse with abundant bright orange sporodochia forming on the carnation leaves. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.7 to 9.3 × 1.3 to 2.9 μm) (n=19). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (27.4 to 35.6 × 3.2 to 4.2 μm) (n=19). These morphological features were consistent with Fusarium oxysporum (Sun et al. 2008; Lombard et al., 2019). To confirm the identification based on these morphological features, the internal transcribed spacer region (ITS) and translation elongation factor1 (TEF) were amplified from the DNA of 3 out of 19 isolates chosen at random respectively using the set primer ITS1/ITS4 and EF1/ EF2 (Sun, S., et al. 2018; Lombard et al., 2019). BLAST analysis revealed that the ITS sequences (OK147619, OK147620, OK147621) had 99% identity to that of F. oxysporum isolate JJF2 (GenBank MN626452) and TEF sequence (OK155999, OK156000, OK156001) had 100% identity to that of F. oxysporum isolate gss100 (GenBank MH341210). A multilocus phylogenetic analysis by Bayesian inference (BI) and maximum likelihood (ML) trees based on ITS and TEF indicated that the pathogen grouped consistently with F. oxysporum. Three out of 19 isolates chosen at random were selected to evaluate pathogenicity. Uninfected healthy A. roxburghii seedlings about 40 day-old planted in sterilized substrates were sprayed with distilled water containing 2 x 106 conidia per ml suspensions as inoculums, and plants sprayed with distilled water alone served as controls. Plants were then incubated at 25°C and 85% relative humidity. Ten plants were inoculated for each isolate. After 10 days, all plants inoculated developed stem rot symptoms, while control plants remained healthy. Cultures of Fusarium spp. were re-isolated only from inoculated plants with the frequency of 100% and re-identified by morphological characteristics as F. oxysporum, fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of F. oxysporum causing stem rot on A. roxburghii seedlings. As F. oxysporum is a devastating pathogenic fungus with a broad host range, measures should be taken in advance to manage stem rot of A. roxburghii.

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First Report of Heterobasidion parviporum (S Group of H. annosum sensu lato) on Tsuga spp. in Asia

Plant Disease ◽

10.1094/pdis.2003.87.8.1007b ◽

2003 ◽

Vol 87 (8) ◽

pp. 1007-1007

Author(s):

Y. C. Dai ◽

K. Korhonen

Keyword(s):

North America ◽

Native Species ◽

Sensu Stricto ◽

Heterobasidion Annosum ◽

Coniferous Forests ◽

Natural Forests ◽

Demarcation Line ◽

First Report ◽

Pinus Spp ◽

And Control

Members of the Heterobasidion annosum (Fr.) Bref. complex are among the most important pathogens in coniferous forests of Europe and North America. Three intersterile groups (P, S, and F) have been found in this complex from Europe (1) and were recently segregated into three species based on intersterility, host preferences, and morphology (4). In a survey of wood-rotting fungi in China in 2002, Heterobasidion spp. were found on Tsuga chinensis (Franch.) Pritz and T. dumosa (D. Don) Eichl. in natural forests from the northern Sichuan Province of southwestern China (32°43′ to 33°11′ N, 103°50′ to 103°53′ E.). Basidiocarps of the fungus were relatively common on decayed wood in roots of dead trees, stumps, and fallen trunks. We collected four basidiocarps (Dai 4045, 4051, 4214, and 4224 in the Institute of Applied Ecology, Chinese Academy of Sciences, IFP) from three stands of mixed coniferous forests and made 40 homokaryotic, single-basidiospore cultures (02046, 02047, 02050, and 02051 in the Finnish Forest Research Institute). Two homokaryons from each basidiocarp were paired with homokaryotic tester strains of European H. annosum (P group), H. parviporum Niemelä & Korhonen (S group), and H. abietinum Niemelä & Korhonen (F group). The pairings showed that the progeny from the four basidiocarps are H. parviporum. The Chinese isolates did not form clamp connections with H. annosum sensu stricto, and a strong demarcation line developed in all these pairings. In contrast, the Chinese isolates formed clamp connections in almost every pairing with European H. parviporum, the clamp connections developed in both sides of the pairings, and no distinct demarcation line was present in most of these pairings. The Chinese homokaryons were also compatible with European H. abietinum, but in most of these pairings, clamp connections were found in the isolate from China but not in the European tester, and a demarcation line was present in most of the pairings. In contrast to H. annosum sensu stricto, the Chinese basidiocarps had smaller pores (4.5 to 6 per mm versus 3 to 4.5 per mm), and a thin tomentum on the pileal surface in contrast to the basidiocarps of H. abietinum. Previously, H. parviporum was found in Asia on coniferous hosts such as Abies, Larix, Picea and Pinus spp. (2). The North American S group of H. annosum sensu lato attacks species of Tsuga spp. in western North America (3), but to our knowledge, this is the first report of H. parviporum on native species of Tsuga spp. outside North America. References: (1) P. Capretti et al. Eur. J. For. Pathol. 20:231, 1990. (2) Y. C. Dai and K. Korhonen. Eur. J. For. Pathol. 29:273, 1999. (3) G. M. Filip and D. J. Morrison. North America. Pages 405–427 in: Heterobasidion annosum. Biology, Ecology, Impact and Control. CAB International, Wallingford, UK, 1998. (4) T. Niemelä and K. Korhonen. Taxonomy of the genus Heterobasidion. Pages 27–33 in: Heterobasidion annosum. Biology, Ecology, Impact and Control. CAB International, Wallingford, UK, 1998.

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