Discoloration Of Vascular Tissue Research Articles

First Report of Stem Canker Disease Caused by Neoscytalidium dimidiatum on Euphrates Poplar in Xinjiang, China.

Euphrates poplar (Populus euphratica Oliv.) constitutes about 61% of the global poplar population, thriving in arid regions of western China (Wu et al. 2023). It plays a crucial role in maintaining ecological balance, securing oasis agriculture, and driving socio-economic progress in the region. During a June 2023 investigation in the P. euphratica forest within the Hotan area of Xinjiang (37°20'21″N, 79°21'15″E), over 12% of the P. euphratica trees displayed branch withering symptoms. The affected trees exhibited cracked bark, trunk decay, darkened coloration, and an eventual black coal-smoke-like appearance. Fungal spores were notably present beneath peeling bark on trunks and main branches. The deep ulcers extended longitudinally into the cambium, leading to tree mortality. In some cases, lateral spread into the sapwood caused dark discoloration of vascular tissue. Twenty diseased branches from various locations were collected and 5-10 mm2 lesions were excised from the edges. These were then surface-disinfected with 75% ethanol for 30 s and 1% sodium hypochlorite for 2 min. After three rinses with sterile distilled water, excess moisture was removed using sterile filter paper, followed by incubating the samples on Potato Dextrose Agar (PDA) medium. Cultures were subsequently grown at 25 ± 1 ℃ under a 12-h photoperiod for three days, thus resulting in the isolation of 25 fungal strains with similar morphological characteristics. All strains displayed rapid colony growth (40 mm/d). On PDA medium, the mycelium initially presented as a white colony, transitioning to an olive-green to greyish color, finally turning dark-grey to black. Colonies generated mycelia that disintegrated into 0- to 1-septate, cylindrical to round, hyaline to brown arthroconidia, occurring singly or in arthric chains, averaging 8.9 ± 2.1 μm × 4.9 ± 1.3 μm, with a length/width ratio of 1.79. Based on morphological characteristics, the isolates were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). Molecular characterization involved amplifying the partial internal transcribed spacer (ITS) region and translation elongation factor 1-α (TEF1-α) and β-tubulin (TUB2) genes using ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and BT2a/BT2b primers (Glass and Donaldson 1995). Sequences, available in GenBank (ITS: PP033096, PP033097, PP033098; TUB2: PP032812, PP032813, PP032814; TEF1-α: PP032815, PP032816, PP032817), exhibited 99-100% identity with the epitype N. dimidiatum Arp2-D (ITS, MK813852; TUB2, MK816354; TEF1-α, MK816355). Phylogenetic analysis, employing maximum likelihood and Bayesian inference on concatenated ITS-TEF1-TUB, was constructed using IQ-Tree and MrBayes3.2.7, revealing isolates clustering within the N. dimidiatum clade. Three isolates (HY01, HY02, and HY05) from different collection points were chosen for pathogenic investigation. Pathogenicity testing on one-year-old healthy P. euphratica seedlings involved removing a 4-mm-diameter bark plug using a cork borer. A 3-day-cultured N. dimidiatum plug of the same size was inoculated, with a blank PDA as control. The wound was covered with moistened sterile absorbent cotton and finally sealed with parafilm for three days. Experiments were repeated thrice. Symptoms manifested by day 2 post-inoculation, resembling the original symptoms by day 7. In the control group, plants remained healthy. N. dimidiatum was exclusively re-isolated from lesions on inoculated stems, confirmed as N. dimidiatum through morphological characteristics and sequence analysis, aligning with Koch's hypothesis. To our knowledge, this is the first report of N. dimidiatum inducing stem canker on P. euphratica in China. This pathogen has been reported on many tree hosts including citrus (Alananbeh et al., 2020), common fig (Güney et al., 2022), dragon fruit (Salunkhe et al., 2023), and Almond (Nouri et al., 2018). Therefore our findings will serve as a warning for authorities to a potential threat in China's P. euphratica and other trees cultivation. Thus, further epidemiological studies are essential for devising effective management strategies.

First report of Fusarium oxysporum f. sp. lactucae Race 1 causing lettuce wilt in Northern Ireland.

In the UK, lettuce is produced both in the field and in greenhouses or polytunnels. In summer 2022, wilt symptoms were observed for the first time on lettuce (cv. Amica) grown in soil in part of a single 0.55 ha greenhouse in County Armagh, Northern Ireland (NI). Initial presentation of symptoms was stunting of plants, followed by wilting and yellowing of lower leaves in approx. 12% of the plants. Orange-brown discoloration of vascular tissue in the tap root of affected plants was also observed. To isolate the causal pathogen, sections (0.5 cm2) of symptomatic vascular tissue from 5 plants were surface sterilized in 70% ethanol for 45 s, washed twice in sterile water and placed on potato dextrose agar (PDA) amended with 20 µg mL-1 chlortetracycline. Plates were incubated at 20°C for 5 days and fungal colonies sub cultured onto PDA. Isolates from all five samples displayed morphology typical of Fusarium oxysporum and were cream to purple in colour with abundant microconidia and occasional macroconidia. DNA was extracted from 5 isolates and part of the translation elongation factor 1- α (EF1-α) gene amplified by PCR and sequenced as described by Taylor et al. (2016). All EF1-α sequences were identical (OQ241898) and matched with F. oxysporum f. sp. lactucae race 1 (MW316853.1, isolate 231274) and race 4 (MK059958.1, isolate IRE1) with 100% sequence identity using BLAST. Isolates were then identified as FOL race 1 (FOL1) using a race-specific PCR assay (Pasquali et al. 2007). Next, pathogenicity and race identity of one isolate (AJ773) was confirmed using a set of differential lettuce cultivars (Gilardi et al. 2017); Costa Rica No. 4 (CR; FOL1 resistant), Banchu Red Fire (BRF; FOL4 resistant) and Gisela (GI; FOL1 / FOL4 susceptible). Plants were inoculated with AJ773 (this study), ATCCMya-3040 (FOL1, Italy; Gilardi et al. 2017) and LANCS1 (FOL4, UK; Taylor et al., 2019). Roots of 16-day-old lettuce plants (8 replicates per cultivar/isolate) were trimmed and soaked in a spore suspension (1 x 106 conidia mL-1) for 10 min before transplanting into compost in 9 cm pots. Control plants for each cultivar were dipped in sterile water. Pots were placed in a glasshouse (25C day, 18C night). Inoculation with AJ773 and FOL1 ATCCMya-3040 resulted in typical symptoms of Fusarium wilt for BRF and GI 12-15 days after inoculation, while for FOL4 LANCS1, wilting was observed in CR and GI. Thirty-two days after inoculation, plants were cut longitudinally and vascular browning was observed in all plants where wilt was present. All uninoculated control plants, CR inoculated with FOL1 ATCCMya-3040 or AJ773, and BRF inoculated with FOL4 LANCS1 remained healthy. These results confirm the identity of isolate AJ773 from NI as FOL1. Koch's postulates were fulfilled by consistent re-isolation of F. oxysporum from BRF and GI plants and identifying as FOL1 using the race-specific PCR. No FOL was re-isolated from control plants of any cultivar. Fusarium wilt in England and the Republic of Ireland first reported by Taylor et al. (2019) was identified as FOL4 and has been confined to indoor lettuce production with further outbreaks caused by the same race. However, FOL1 was recently identified in Norway in a soil-grown glasshouse crop (Herrero et al. 2021). The presence of both FOL1 and FOL4 in neighbouring countries within the UK is a serious risk to lettuce production and is of particular importance to growers who rely on knowledge of cultivar resistance to specific FOL races to make decisions on which variety to plant.

First report of Fusarium solani causing wilt disease on Gleditsia sinensis in Tianjin, China.

The Gleditsia sinensis Lam. widely grown in China is a perennial plant with medicinal properties (Zhang et al. 2016). Since 2019, the leaves of G. sinensis have exhibited yellowing and wilting, and the plants have gradually become stunted and dead in Taifeng park of Binhai New Area in Tianjin (39.02° N; 117.65° E). In this park, there are two types of G. sinensis, one is with round branch thorns, the other is with flat branch thorns. The G. sinensis with round branch thorns did not grow well and almost all plants had disease symptoms. The samples were collected on October, 2021 and deposited in Plant Disease Laboratory of Tianjin Agricultural University under accession no. PATAU211018. The disease symptoms consisted of foliage wilt (Figure 1A), plant drying and vascular tissue discoloration (Figure 1B). The stem sections from different plants were surface-disinfested in 0.6% NaClO, wiped with 75% ethanol and rinsed with sterile water. Thirty tissue samples were placed on Potato Dextrose Agar (PDA) medium and cultured at 28℃ for 7 days (Uppala et al. 2013). Thirty fungal isolates with the same morphological characteristics were obtained from the samples. Five representative isolates (PATAU211018-05, PATAU211018-07, PATAU211018-10, PATAU211018-12 and PATAU211018-21) were collected and purified using the single-spore method (Li et al. 2022). Colonies of the five isolates on PDA grew in a circular shape and showed abundant white densely fluffy aerial mycelium (Figure 1C). Morphological characteristics included septate and hyaline hyphae, long cylindrical monophialides (Figure 1D), macroconidia (Figure 1E) and microconidia (Figure 1F). Macroconidia were falcate, 2-5-septate, hyaline, 18-40 × 4-6 μm (n = 50). Microconidia were hyaline, oblong, 0-1-septate, 5-14 × 2-6 μm (n = 50). These morphological characteristics were consistent with the description of Fusarium solani. (Chitrampalam et al. 2018). PATAU211018-12 was randomly chosen for molecular analysis as the representative isolate given the similarity of these isolates. For further identification, the genomic DNA of isolate PATAU211018-12 was extracted. The fragments of internal transcribed spacer (ITS), translation elongation factor 1α (EF1α) gene and RNA polymerase II subunit (RPB2) were amplified and sequenced (O'Donnell et al. 2008; Carbone et al. 1999). The sequences of ITS, EF1α, and RPB2 of PATAU211018-12 were deposited in GenBank under the accession no. of OP735578, ON630412 and OP746032, respectively. Phylogenetic trees were constructed in MAGA X (Kumar et al. 2018) using the neighbor-joining (NJ) method based on the concatenated sequences of ITS, EF1α, and RPB2 (Figure 2). The isolate (PATAU211018-12) grouped with F. solani (JS-169) with a bootstrap value of 100 in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolate is F. solani. Pathogenicity tests were carried out on one-year-old G. sinensis seedlings with round branch thorns (n=6). The F. solani isolate PATAU211018-12 was cultured in Potato Dextrose Broth (PDB) at 28°C on a shaker at 150 rpm for 5 days. Mycelia were filtered through four layers of sterilized lens paper and the conidia were obtained for pathogenicity tests. G. sinensis was infected by F. solani through root soaking method. The roots were inoculated by dipping in conidial suspension with the concentration of 107 conidia/mL for 30 minutes. Control plants (n=6) were treated with distilled water. Plants were in pots indoors at 25℃. At 20 days after inoculation, the leaves of inoculated plants were chlorotic and wilted (Figure 1G), symptoms similar to those observed in the park. In contrast, the leaves of control plants were symptomless (Figure 1H). The pathogenicity assay was repeated three times. The fungal isolate was re-isolated from the disease tissues and verified as F. solani based on morphology and molecular character (ITS, EF1α and RPB2). F. solani has been reported as pathogens on many plants, such as Eriobotrya japonica (Wu et al. 2021), Fragaria × ananassa (Pastrana et al. 2014), Gastrodia elata (Li et al. 2022) and Hedysarum boreale (Uppala et al. 2013). To our knowledge, this is the first report of F. solani causing disease on G. sinensis in China. Identification of F. solani as a disease agent in G. sinensis will assist in disease management for this important tree crop.

First report of Lasiodiplodia theobromae causing stem canker of pomegranate (Punica granatum L.) in India.

In March 2022, cankers and lesions appeared on the branches of 2-3-year-old pomegranate plants grown in four orchards of Hanumangarh, Rajasthan, India. The disease incidence ranged from 5-15%. Field symptoms such as dark brown lesions on one side of the branches, cracked lesions, vascular tissue discoloration and drooping of the plants were noticed. To identify the causative agent, 2 diseased branch samples, showing typical symptoms collected from each orchard 25-30 km apart. The samples were washed with distilled water and small sections of tissue were excised from both symptomatic and asymptomatic areas using a sterile scalpel. Sections were surface sterilized with 1% sodium hypochlorite for 30 sec and 70% ethanol for 2 min followed by rinsing with sterilized water thrice. Sterile sections were dried on sterile filter paper and then transferred onto potato dextrose agar (PDA) amended with streptomycin (100 mgL-1) and incubated at 24±1°C in the dark. Samples (n=5) collected from different orchards produced similar colonies, with greyish white aerial mycelia, which became dark black after 5-7 days. The morphological characteristics of all isolates were observed under microscope. Immature conidia (6.3±1.05*14.7±0.98 μm: average of 50 measurements) were single celled, hyaline, ellipsoid or ovoid, apex rounded and truncated at the base while the matured conidia (8.4±1.41*15.3±1.17 μm: average of 50 measurements) had two cells with dark septa. The conidial morphology of all isolates was in accordance with Lasiodiplodia sp. (Alves et al; 2008) therefore, one representative isolate (HSC-1) was used for molecular identification at species level. Three loci viz., ITS, EF1-a and β tubulin of fungal genomic DNA were PCR amplified using ITS-1/4, EF-F/R and TUB-2A/2B primers, respectively. The amplicons were sequenced and deposited in GenBank, NCBI database with accession no. ON598885 (ITS), ON605203 (EF) and ON605204 (TUB). BLASTn analysis showed similarity with the sequences of Lasiodiplodia theobromae isolates: ITS showed 100% with MK530071.1 (492 bases), EF 99.77% with MT975688.1 (436 bases) and BT 99.76% with MW287586.1 (422 bases). Phylogenetic analysis using Neighbour Joining method revealed close association among L. theobromae isolates. Thus, causative agent associated with stem canker of pomegranate was confirmed as L. theobromae. Further, the same isolate was used for pathogenicity tests on 1-year-old pomegranate plants (n=6). Briefly, 2 cm wound was created in the main stem with a sterile scalpel and a same-size mycelial plug was placed in the wound and wrapped with parafilm. Six plants that were wrapped with uncultured PDA served as control. The inoculated plants were maintained at 26°C and 65-70% RH in a polyhouse. After 4 days parafilm was removed from all plants. The experiment was repeated twice. Inoculated plants produced lesions (0.7 x 5.5 cm; average of 6 measurements) similar to field symptoms after 10-15 days and no such symptoms developed on control plants. The difference between control and inoculated plants was statistically significant (p=0.0001). The fungus was re-isolated from symptomatic tissue and colonies were morphologically similar to HSC-1, thus fulfilling the Koch's postulates. The fungus, L. theobromae causes stem canker and dieback on different host plants and is mainly distributed in tropical and subtropical regions and has been reported on pomegranate from Florida (Xavier et al 2017). To the best of our knowledge, this is the first report of L. theobromae causing stem canker of pomegranate in India.

First report of dieback on fig (Ficus carica L.) caused by Lasiodiplodia theobromae in North Al-Batinah governorate of Oman.

Fig (Ficus carica L.) is widely grown in Oman mainly for its nutritional value and health benefits. In May 2020, survey was conducted for fig trees which were showing symptoms of decline in three farms where more than 1500 fig trees were grown. The incidence of the disease was 3 to 4 % of fig trees showing symptoms of dieback of the twigs, death of entire branch and discoloration of vascular tissues were observed in Sohar, North Al-Batinah governorate of Oman. The disease severity was range between 25 to 40 % in most of the infected tree. The death of severely affected trees was observed in about 3 % of the orchards. Symptomatic samples were collected and isolation of the causal agent was performed. Infected tissues were cut into small pieces and surface-sterilized with 1 % NaOCl for 3 min. The tissues were rinsed with sterile distilled water 3-4 times, blotted dry on sterile filter paper and placed on potato dextrose agar (PDA) medium under aseptic conditions. After 2-3 days of incubation of plates at 27°C, pure culture of the fungus was obtained by hyphal tip isolation technique. Two identical isolates were grown in the PDA medium. The fungal colony was dark grey to black colored on PDA medium and the fungus produced dense aerial mycelium and numerous dark colored pycnidia in 25 days at ambient temperature range between 23-25 ℃ and nature photoperiod. Pycnidia were raptured and 20 emerged conidia were measured. The mature conidia were 1-septate, brown, ellipsoid, with thick cell wall and having longitudinal striation. The average measurement of the length of 20 conidia was 22.5-25 (24.9 µm) however the width measured was 12.5-15 (15 µm). To identify the fungus, DNA was extracted from the mycelium using phenol-chloroform-isoamyl alcohol method, following Al-Mahmooli et al. (2015). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and translation elongation factor 1-α (TEF) gene were amplified using ITS-4 and ITS-5 primers for ITS (White et al. 1990), EF1-983F and EF1-2218R and EF1-728F and EF1-986R primers for TEF (Alves et al. 2008), respectively and the amplified products were sequenced. BLASTn analysis of the ITS sequence of the fungal isolate revealed 100% identity with reference sequences of Lasiodiplodia theobromae (Pat.) Griffon & Maubl (MT075438.1). The sequence of TEF gene was 99% identical to the sequences of L. theobromae (XM_035519539.1 and MN461169.1). The nucleotide sequences were submitted to GenBank and accessioned with (MW590660) for ITS and (MZ159970, MZ159971, OM654917) for TEF gene. For phylogenetic analysis, we constructed a combined ITS-TEF dataset, following (Alves et. 2008). Using Maximum Likelihood phylogenetic analysis of the combined ITS-TEF dataset, our specimen falls in a clade formed by the members of L. theobromae (Supplementary materials). Pathogenicity of the fungus of three replicates (three fig seedlings) was established by artificial inoculation of 2- to 3-month-old potted fig seedlings with the fungus by making pin prick injuries on the stem at 5 cm above the soil level with a sterile inoculation needle, placing mycelial discs containing spores and pycnidia (15 × 15 mm) from 20-days old colonies grown on PDA on the injured surface and wrapping with parafilm which removed 48 hrs after establishing of infection. The three inoculated plants showed withering and drying of leaves 14 days after inoculation (DAI) begins near to the inoculation site expend to the younger leaves. Eventually, seedlings were showing dieback and death of entire seedling occurs in 25-28 DAI. The mock-inoculated plants remained symptomless. The fungus re-isolated from the diseased plants was confirmed by sequencing of ITS regions. The prevalence of dieback could be a potential threat to cultivation of fig in Oman. To the best of our knowledge, this is the first report of dieback disease caused by L. theobromae on fig in Oman.

Root Rot of Angelica sinensis Caused by Clonostachys rosea and Fusarium acuminatum in China.

Angelica sinensis is a perennial plant in the Apiaceae and a source of one of the most important traditional medicines in China. This medicinal herb has been mainly cultivated in Gansu, Sichuan, Yunnan, Shanxi, Hubei, and Qinghai Provinces. In June 2020, angelica plants in a field in the village of Sada, Minhe County, Haidong city, in Qinghai Province (102°83'00.00″E, 36°31'93.00″N, elevation:2437 m), displayed symptoms of yellowing, stunting, root rot, and wilting（Fig. S1）. Severe brown discoloration of vascular tissue in the stems of infected plants was observed. Root rot seriously affected the quality and yield of A. sinensis. The incidence of root rot in the fields reached 15% in 2020 and 18% in 2021. Fungi were isolated from diseased roots using a single spore method. The identity of isolates was confirmed by sequencing the nuclear ribosomal internal transcribed spacer (ITS) using the primers ITS1/ITS4 (White et al, 1990) and the translation elongation factor 1-alpha (TEF1-α) gene using the primers EF-1/EF-2 (O'Donnell et al. 1998). BLAST analysis of the ITS and TEF1-α sequences of isolates DG-1, DG-1-1, DG-1-2, DG-2, DG-2-1, DG-5, and DG-5-1 in the NCBI database showed 99% to 100 % nucleotide identity to those of Clonostachys rosea (NCBI accession Nos. MZ424803-MZ424809 (ITS), and MZ451383-MZ451389 (TEF1-α)). BLAST analysis of the ITS and TEF1-α sequences of isolates, DG-3, DG-3-1, and DG-4 in the NCBI database produced matches of 99.1% or better to Fusarium acuminatum (NCBI accession Nos. MZ424810-MZ424812 (ITS), and MZ441148-MZ441150 (TEF1-α)). Conidia of C. rosea were hyaline, round or oval, aseptate, with an average length and width of 6.5-8.5×2.5-3.5 μm. Chlamydospores were round or oval, with an average length and width of 9.9-16.2×8.1-13.6 μm. Conidiophores of C. rosea were hyaline, septate, Verticillium-like, conidiogenous cells were slender and cylindrical. Macroconidia of F. acuminatum were hyaline, falciform, 0-5 septate, with an average length and width of 22.5-39.5 × 2.5-4.5 μm. Microconidia were rare. Chlamydospores were oval, with an average length and width of 9.5-14.3×8.1-11.8 μm. Conidiophores of F. acuminatum were hyaline, septate, branched (Fig. S2). A pathogenicity test was performed by inoculating a conidial suspension of 1×105 per ml in 0.025% Tween 20 onto roots of A. sinensis. Inoculated roots were incubated in a humid and dark chamber at 26°C for 24 h, and then with 12 h light/12 h dark for six days. The pathogenicity assay was repeated three times for each isolate. Root rot was observed seven days after inoculation and mycelia were observed on the surface of the lesions (Fig. S1). Two isolates were recovered from the lesions and named DG-2-D and DG-3-1-D. The colonial morphology of these two isolates and the original isolates DG-2 and DG-3-1 was identical after six days of inoculation on PDA (potato dextrose agar) (Fig. S2). The two isolates recovered were identified as C. rosea and F. acuminatum, respectively, by amplifying and sequencing a portion of the TEF1-α gene. To our knowledge, this is the first report of C. rosea causing root rot of A. sinensis in China. Fusarium acuminatum was reported to cause root rot of A. sinensis in Gansu province, China in 2021 (Niu et al. 2021). Clonostachys rosea has been previously reported to cause root rot of soybean in the United States (Bienapfl et al. 2012). The disease must be considered in existing management practices, and our findings provide a foundation for management this disease.

First Report of Pseudophaeomoniella oleae Causing Wood Streaking and Decay on Olive Trees in Greece.

A survey was conducted in olive orchards (Olea europaea L., cv Koroneiki) showing severe decline in Milatos and Ierapetra (Lasithi, Crete, Greece) in November 2017 and January 2019, respectively. Diseased trees exhibited wilting, yellowing of leaves, twig and branch dieback, and internal discoloration of vascular tissue. Insect infestations were commonly associated with these symptoms. A yeast-like fungus was consistently isolated from discolored vessels previously surface-disinfested with 95% ethanol, on acidified potato dextrose agar (APDA). The fungus yielded several dark brown to black, globose to oval pycnidia with dimensions 120-330 × 90-300 μm (average 179.6 × 143.9 μm), after 25 days of growth on APDA. Emerging colonies were transferred to new PDA and their growth rate was 2.21 mm/day at 24 °C in the dark. The sparse aerial mycelium was initially white, turning into beige-pinkish in the centre after 21 days of growth on PDA. Microscopic observations revealed hyaline, smooth, ampulliform conidiophores, bearing conidia solitary or in slimy heads. Conidia were 1-celled, hyaline, smooth, subcylindrical with obtuse ends, 1.25-5.75 × 0.75-2.00 μm (average 3.12 × 1.16 μm) in size. Light to dark brown pycnidia, semi-immersed in PDA, with dimensions 150-490 × 90-320 μm (average 297.7 × 231.0 μm) were evident in 3-week-old cultures. Colony morphology and microscopic features were similar to Pseudophaeomoniella oleae (Crous et al. 2015). DNA from two representative isolates (EML1 and DRAGVR1) was extracted and their internal transcribed spacer region (ITS) of ribosomal DNA, actin (ACT) and translation elongation factor 1-alpha (TEF1-α) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. PCR products were sequenced and deposited in GenBank (accession Nos. MZ854242-MZ854243, OK143463-OK143464 and OK143465-OK143466). BLAST search revealed high similarity to GenBank sequences of the ex-type strain of P. oleae for ITS (≥99.50%, NR_137966.1 and KP635972.2), ACT (100%, KP635974.1), and TEF-1a (98.32%, KP635968.1). Based on morphology and phylogenetic analysis of the ITS region, the fungus was identified as P. oleae. Ten 3-year-old olive trees of each of the cvs Amfissis and Koroneiki were artificially inoculated by drilling a 3-mm-diameter hole into the trunk and injecting 50 μl of a 1 × 107 ml-1 conidia suspension of the isolate DRAGVR1 (Markakis et al. 2017). Another ten trees of each cv treated similarly with sterilized distilled water served as controls. Potted trees were kept under ambient conditions. Fourteen months post inoculation, longitudinal and transverse sections of inoculated trunks revealed wood discoloration extending above and below the inoculation point in both cultivars, whereas no leaf symptoms were observed. P. oleae was steadily re-isolated from symptomatic wood tissue and identified by colony morphology. Neither symptoms nor positive isolations were observed in controls. To our knowledge, this is the first report of wood streaking and decay caused by P. oleae in olive trees in Greece. Although the fungus has been isolated previously from decayed olive trees showing vascular wilt in Italy (Crous et al. 2015), this is the first experimental evidence of the pathogenic potential of the species on olive trees worldwide. This disease could potentially be an increasing problem in olive tree growing areas and result in severe crop losses. Hence, effective management practices should be investigated and applied.

First Report of Fusarium oxysporum f. sp. lactucae Race 1 Causing Fusarium Wilt of Lettuce in Norway.

Lettuce (Lactuca sativa L.) is produced in Norway both in field and greenhouses. In Norway, greenhouse lettuce is one of the most important vegetables grown year-round. In winter 2018, wilting symptoms were observed on soil-grown lettuce of the cultivar Frillice in a greenhouse in south east Norway (Buskerud county). Affected plants showed stunted growth, wilting of outer leaves, and brownish discoloration of vascular tissues of taproots and crowns. According to the producer, the disease led to an estimated 10% of yield losses. Fungal isolates were obtained from crowns and roots of diseased plants collected from the greenhouse in 2018 and 2019. Two single spore isolates, 231274 from 2018 and 231725 from 2019, were used in further studies. The isolates were incubated on synthetic nutrient-poor agar (SNA) at 18-20 ⁰C, and a 12 hours dark, 12 hours UV light cycle. Isolate 231274 produced abundant macro- and microconidia characteristics of Fusarium oxysporum while macroconidia were never observed in isolate 231725. On potato dextrose agar (PDA), colonies of isolate 231274 were purple in color and colonies of isolate 231725 were pinkish with abundant aerial mycelium. For PCR-assay, DNA from mycelia was extracted using Easy-DNA kit (Invitrogen). A portion of the translation elongation factor 1-α (EF1-α) gene was amplified using primers F-728F (Carbone and Kohn. 1999) and EF2 (O'Donnell et al. 1998) as described by Aas et al. 2018. Blast analysis of both sequences (accession no. MW316853 for 231274 and MW316854 for 231275) obtained a 99% homology with the sequence of Fusarium oxysporum f.sp. lactucae (FOL) race 1 strain S1 (accession no. DQ837657)(Mbofung et al. 2007). Both isolates were identified as race 1 by using specific primers Hani3' and Hanilatt3rev (Pasquali et al. 2007) as described by Cabral et al. 2014. To complete Koch's postulate, lettuce plants of the cultivar Frillice were used. Race identity was confirmed using the differential lettuce cultivars Costa Rica No.4 (resistant to FOL race 1), Banchu Red Fire (resistant to FOL races 2 and 4) and Romana Romabella (resistant to FOL races 1 and 2) (Gilardi et al. 2017) provided by the breeding company Rijk Zwaan (De Lier, The Netherlands). For inoculation, roots of six 2-weeks old seedlings per cultivar were dipped in a spore suspension (1 x 106 CFU/ml) for 1 min, while controls were dipped in distilled water. Seedlings were planted in 250 ml pots containing fertilized potting substrate, and were placed in a greenhouse with temperature ranging from 15 to 35 ⁰C and an average of 23 ⁰C. After 10 days reduced growth was observed in cultivars Frillice and Banchu Red Fire for both fungal isolates. After 25 days wilting was observed in both cultivars. Affected plants presented discoloration of vascular tissue. No difference in growth was observed between cultivars Romana Romabella and Costa Rica No. 4 and their respective controls. FOL was re-isolated from all inoculated cultivars but not from controls. The colony patterns of the recovered isolates were the same than those of the isolates used for inoculation. These results confirm that the isolate belongs to race 1. Greenhouse lettuce in Norway is mainly produced in hydroponics. FOL is here reported to cause damages in soil- grown lettuce. Nevertheless FOL in hydroponic systems has been reported in Japan (Fujinaga et al. 2003) and Thailand (Thongkamngam and Jaenaksorn 2017). Thus, the possibility of infections in hydroponics remain a big concern for lettuce production in Norway.

Molecular evidence of the avocado defense response to Fusarium kuroshium infection: a deep transcriptome analysis using RNA-Seq.

Fusarium kuroshium is a novel member of the Ambrosia Fusarium Clade (AFC) that has been recognized as one of the symbionts of the invasive Kuroshio shot hole borer, an Asian ambrosia beetle. This complex is considered the causal agent of Fusarium dieback, a disease that has severely threatened natural forests, landscape trees, and avocado orchards in the last 8 years. Despite the interest in this species, the molecular responses of both the host and F. kuroshium during the infection process and disease establishment remain unknown. In this work, we established an in vitro pathosystem using Hass avocado stems inoculated with F. kuroshium to investigate differential gene expression at 1, 4, 7 and 14 days post-inoculation. RNA-seq technology allowed us to obtain data from both the plant and the fungus, and the sequences obtained from both organisms were analyzed independently. The pathosystem established was able to mimic Fusarium dieback symptoms, such as carbohydrate exudation, necrosis, and vascular tissue discoloration. The results provide interesting evidence regarding the genes that may play roles in the avocado defense response to Fusarium dieback disease. The avocado data set comprised a coding sequence collection of 51,379 UniGenes, from which 2,403 (4.67%) were identified as differentially expressed. The global expression analysis showed that F. kuroshium responsive UniGenes can be clustered into six groups according to their expression profiles. The biologically relevant functional categories that were identified included photosynthesis as well as responses to stress, hormones, abscisic acid, and water deprivation. Additionally, processes such as oxidation-reduction, organization and biogenesis of the cell wall and polysaccharide metabolism were detected. Moreover, we identified orthologues of nucleotide-binding leucine-rich receptors, and their possible action mode was analyzed. In F. kuroshium, we identified 57 differentially expressed genes. Interestingly, the alcohol metabolic process biological category had the highest number of upregulated genes, and the enzyme group in this category may play an important role in the mechanisms of secondary metabolite detoxification. Hydrolytic enzymes, such as endoglucanases and a pectate lyase, were also identified, as well as some proteases. In conclusion, our research was conducted mainly to explain how the vascular tissue of a recognized host of the ambrosia complex responds during F. kuroshium infection since Fusarium dieback is an ambrosia beetle-vectored disease and many variables facilitate its establishment.

Phylogeny and pathogenicity of soilborne fungi associated with wilt disease complex of tomatillo (Physalis ixocarpa) in northern Sinaloa, Mexico

Wilt disease complex is one of the most important diseases of tomatillo (Physalis ixocarpa) in the production areas of Mexico. Disease symptoms include wilting, poor growth, discoloration of vascular tissues, root rot, and death of plants. The aims of this study were to identify the fungi associated with wilt disease complex of tomatillo by the combination of phylogenetic analyses and morphological characterization, as well as to determine their pathogenicity and virulence on tomatillo seedlings. A total of 88 fungal isolates were obtained from symptomatic plants from 19 tomatillo fields distributed in northern Sinaloa, Mexico. Subsequently, a subset of 37 isolates representing the range of geographic origin was selected for further morphological and molecular characterization as well as pathogenicity tests. Phylogenetic analyses using Maximum Likelihood were used to identify 15 isolates of Rhizoctonia (ITS sequence data), 14 isolates of Fusarium (EF-1α sequence data), five isolates of Macrophomina (ITS, EF-1α, BT, and ACT sequence dataset) and three isolates of Neocosmospora (EF-1α sequence data) to species level. Pathogenicity tests were performed on tomatillo seedlings (cv. Gran Esmeralda) under greenhouse conditions. Phylogenetic analyses of 37 fungal isolates allowed the identification of Rhizoctonia solani AG 4-HGI (40.5%), Fusarium oxysporum (29.8%), Macrophomina phaseolina (13.5%), F. nygamai (8.1%) and Neocosmospora falciformis (8.1%). All fungal species were found to be pathogenic on tomatillo seedlings but a significant difference in disease severity was observed. To our knowledge, F. nygamai, M. phaseolina and N. falciformis were recorded for the first time infecting tomatillo in Mexico and worldwide.

Occurrence of Root Rot Caused by Fusarium fujikuroi on Soybean (Glycine max) in the Central Eastern Regions, China

Occurrence of Root Rot Caused by <i>Fusarium fujikuroi</i> on Soybean (<i>Glycine max</i>) in the Central Eastern Regions, China

First Report of Crown and Root Rot Caused by Fusarium oxysporum on Sweet Pepper (Capsicum annuum) in Italy

During August 2018, sweet pepper (Capsicum annuum L.) cv. Cuneo, grown in 3,000 m² of commercial greenhouses near Cuneo in Piedmont, northern Italy, exhibited symptoms of reduced growth and sudden wilting. Crown and roots were severely affected, showing a brown necrosis and slight rot. No internal discoloration of vascular tissues was observed. Eventually, affected plants collapsed. Symptoms developed on approximately 10% of the plants 3 months after transplanting. Tissue fragments of 1 mm² were excised from the crown and roots of 10 symptomatic plants, dipped in a 1% sodium hypochlorite solution, washed in sterile water, and placed on potato dextrose agar. Fungal colonies were isolated from all samples incubated under constant fluorescent light at 22 ± 1°C for 6 days. Thirty-day-old single-spore cultures of four selected isolates (FOC1, FOC2, FOC3, and FOC4) grown on carnation leaf agar generated short monophialides with unicellular, ovoid-elliptical microconidia, measuring 5.6 to 13.5 × 2.5 to 3.5 µm. Sparse macroconidia were 3-septate, slightly curved, with a foot-shaped basal cell and a short apical cell, and measured 19.4 to 38.3 × 2.9 to 4.5 µm. Chlamydospores were terminal and intercalary, single, in pairs, clusters, or in chains, and measured 6.7 to 10.6 µm in diameter. Such characteristics are typical of Fusarium oxysporum (Leslie and Summerell 2006). DNA was extracted from the four isolates and molecular identification was carried out by sequencing the portions of the elongation factor-1 alpha (TEF-1α) gene (O’Donnell et al. 1998) and the intergenic spacer (IGS) region (GenBank accession nos. MK061116–MK061119 for TEF-1α and MK061120–MK061123 for IGS). A BLASTn search showed that the TEF-1α sequences (625 bp) were 99 to 100% similar to the corresponding sequences HG916993–HG916995 of F. oxysporum f. sp. radicis-capsici from sweet pepper (Lomas-Cano et al. 2014), while the IGS sequences (1187–1274 bp) were 99% similar to that of F. oxysporum (FJ985621). The four isolates were used for pathogenicity tests conducted in 2-liter pots filled with steamed peat substrate in a greenhouse at 25 to 28°C. Twenty-day-old sweet pepper plants (cv. Cuneo) at the three true leaf stage were inoculated by dipping roots in a conidial suspension (1 × 10⁶ conidia/ml) obtained from a 10-day-culture in potato dextrose broth. Twenty plants (5 plants/pot) per treatment were used in two repeated trials for each isolate, and 20 noninoculated plants were used as controls. First wilt symptoms developed 8 to 10 days after inoculation, and 70 to 100% of inoculated plants were dead 15 to 20 days after inoculation, showing crown and root rot. Tomato plants (Solanum lycopersicum) cv. Cuore di bue, inoculated with the four isolates under the same conditions, did not develop symptoms after 50 to 60 days. All noninoculated control plants remained healthy. A fungus morphologically identified as F. oxysporum was consistently reisolated from all the symptomatic pepper plants and confirmed by the TEF-1α and the IGS analysis. To our knowledge, this is the first report of F. oxysporum on sweet pepper in Italy. Although F. oxysporum f. sp. capsici is reported on sweet pepper (Pernezny et al. 2003), symptoms observed in this study were very similar to those caused by F. oxysporum f. sp. radicis-capsici described by Lomas-Cano et al. (2014, 2016) on sweet pepper seedlings in Spain. The disease, although limited at present in the greenhouses surveyed in northern Italy, could expand to other areas where sweet pepper is grown intensively.

Identification and Pathogenicity of Fungal Species Associated with Canker Diseases of Pistachio in California.

A survey was conducted during 2015 and 2016 in pistachio orchards throughout the San Joaquin Valley of California to investigate the occurrence of canker diseases and identify the pathogens involved. Cankers and dieback symptoms were observed mainly in orchards aged >15 years. Symptoms of canker diseases included brown to dark brown discoloration of vascular tissues, wood necrosis, and branch dieback. In total, 58 fungal isolates were obtained from cankers and identified based on multilocus phylogenetic analyses (internal transcribed spacer, glyceraldehyde 3-phosphate dehydrogenase, β-tubulin, calmodulin, actin 1, and translation elongation factor 1α) representing 11 fungal species: Colletotrichum karstii, Cytospora californica, Cytospora joaquinensis, Cytospora parapistaciae, Cytospora pistaciae, Diaporthe ambigua, Didymella glomerata, Diplodia mutila, Neofusicoccum mediterraneum, Phaeoacremonium canadense, and Schizophyllum commune. Pathogenicity tests conducted in the main pistachio cultivars Kerman, Golden Hills, and Lost Hills using the mycelium-plug method indicated that all fungal species were pathogenic to Pistacia vera. All species tested caused cankers in pistachio branches, although virulence among species varied from high to moderate. Overall, N. mediterraneum and Cytospora spp. were the most widespread and virulent species associated with canker diseases of pistachio in California.

Wilt of Shantung Maple Caused by Verticillium dahliae in China

Shantung maple (Acer truncatum Bunge) is an important economic plant, widely grown in Northern China. The seed oils contain nervonic acid which can reduce the risk of many mental illnesses (Hu et al. 2017). In June 2016, some trees showed symptoms of wilting, leaf fall, branch and twig dieback, and discoloration of vascular tissues in Jining, Shandong. The leaves of the diseased trees only showed dehydration without yellowing. The xylem also became black. The incidence of diseased plants was more than 30% on two farms (total of 4882 trees) where eggplants and soybeans were previously planted. As we know, eggplants tend to be infected by Verticillium dahliae in China (Zhu et al. 2006). To identify the pathogen, five stems (each approximately 70 mm in diameter and 10 cm long) sampled from five symptomatic plants were flushed with tap water for 30 min, surface-sterilized with 75% ethanol for 5 min, rinsed in sterilized water three times and dried. The discolored vascular tissues were cut into 0.5 cm long seg...

First Report of Root Rot and Vine Decline of Melon Caused by Monosporascus cannonballus in Greece

In June 2017, a severe vine decline of the netted melon (Cucumis melo L.) hybrids Global F1 and Lavigal was observed in a greenhouse of Messara valley, Heraklion, Crete, Greece. Diseased plants exhibited wilting, leaf chlorosis, yellowing and vine collapse. Root rot and necrosis, foot rot and to a lesser extent vascular tissue discoloration at the lower part of the stem were also observed. Numerous perithecia embedded in the root cortex, containing asci and ascospores typical of Monosporascus cannonballus Pollack & Uecker (1974) were observed. Disease incidence in the greenhouse was estimated at 100 and 78% for Global F1 and Lavigal, respectively. The fungus was isolated from the root lesions and the discolored vessels of the basal part of the stem, previously surface-sterilized with 1.5% sodium hypochlolrite for 1 min, on potato dextrose agar (PDA) amended with 0.5 g/l streptomycin sulfate. To obtain single hyphae isolates, hyphal tips were transferred onto new PDA plates and the growth rate of the fungu...

First Report of Fusarium Wilt on Cavendish Bananas, Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (VCG 01213/16), in Vietnam

Fusarium wilt of banana, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is a major constraint to banana production worldwide. A strain of the fungus that affects Cavendish and other dessert bananas in the tropics, called Foc tropical race 4 (TR4; VCG 01213/16), has been confined to five Asian countries (Indonesia, Malaysia, Philippines, mainland China, and Taiwan) for more than three decades. Cavendish production is now being expanded in Asia to Laos, Myanmar, and Vietnam, where local varieties still dominate the market. This is owing to an increase in Cavendish banana consumption and a decline in areas of production caused by Foc TR4 in China. The expansion of Cavendish production in Southeast Asia, however, has increased the risk of Foc TR4 being introduced into new countries. Leaf-yellowing symptoms typical of banana Fusarium wilt were observed in three provinces in northern Vietnam along the Red River that originates in the Yunnan province of China, namely Hanoi (October 2014), Hung Yen (October 2015), and Lao Cai (October 2015). When cut open, pseudostems displayed a dark-red to brown discoloration of vascular tissue, and the inner rhizome revealed a ring of yellow-brown staining. Samples of symptomatic strands from pseudostems were collected to identify the causal agent by morphological, molecular, and vegetative compatibility group (VCG) identification. The samples were first plated onto potato dextrose agar (PDA) containing streptomycin for fungal isolation. The cultures were then purified, single-spored, and maintained at the culture collection of the Department of Plant Pathology at Stellenbosch University in South Africa. Eight Fusarium isolates obtained were identified to species level on PDA and carnation leaf agar plates according to their cultural and spore morphology (Nelson et al. 1983). For molecular identification, total DNA was extracted and amplified with Foc TR4-specific primers (Dita et al. 2010) and Foc race 4-specific primers (Lin et al. 2009). VCG testing was performed according to the method of Puhalla (1985) by pairing nit-1 and nit-3 mutants of the isolates from Vietnam with nit-M testers of VCGs 01213, 01216, and 01213/16. Pathogenicity testing was performed by replanting Cavendish tissue culture plantlets at the six-leaf stage in sand mixed with corn powder, which was colonized by the individual Fusarium isolates from Vietnam, at a ratio of 19:1. Control plants were treated with sterile distilled water. The identity of the Vietnamese isolates was confirmed as F. oxysporum, whereas PCR and VCG analysis characterized six of them as VCG 01213/16 strains. After 6 weeks, plants inoculated with the VCG 01213/16 isolates produced typical symptoms of Fusarium wilt, so Koch's postulates were completed. The occurrence of Foc TR4 in Vietnam can have a significant economic impact to agricultural production, because investments were made to replace citrus, longan, and corn fields with Cavendish plantations. Foc TR4 also poses a threat to local banana varieties, and these varieties should therefore be evaluated for resistance to the fungus. The potential spread of Foc TR4 in Vietnam should be minimized by surveillance, public awareness campaigns, risk assessment, and containment efforts.

First Report of Neofusicoccum algeriense Causing Dieback of Red Raspberry in Mexico

During 2014 and 2015, severe symptoms of dieback were observed in a commercial orchard of red raspberry (Rubus idaeus L.) cv. Heritage in Tangancicuaro, Michoacan, Mexico. Symptoms consisted of brown cankers and dieback on canes, accompanied by reddening and wilting of foliage, at an incidence of 5%. Internal symptoms included a brown discoloration of vascular tissues. A total of 30 stem pieces (approximately 20 cm in length) were collected from 15 diseased plants. Sixty colonies grown on potato dextrose agar (PDA) at 25oC developed a white, aerial mycelium that became olivaceous gray at the surface after 5 days. Pycnidia were produced after 2-3 weeks on autoclaved pine needles placed on water agar and were solitary or aggregated, dark brown to black, uniloculate, of 320 to 417 μm in diameter. Conidiophores were hyaline, cylindrical, smooth, thin walled, and septate. Conidia were hyaline, fusiform, base subtruncate to bluntly rounded, smooth, thin walled, aseptate, of (13-) 16 to 19 (-21) x (4-) 4.5 to 5....

Zebra chip disease decreases tuber (Solanum tuberosum L.) protein content by attenuating protease inhibitor levels and increasing protease activities.

Zebra chip disease of potato decreases protease inhibitor levels resulting in enhanced serine-type protease activity, decreased protein content and altered protein profiles of fully mature tubers. Zebra-chip (ZC), caused by Candidatus Liberibacter solanacearum (CLso), is a relatively new disease of potato that negatively affects growth, yield, propagation potential, and fresh and process qualities of tubers. Diseased plants produce tubers with characteristic brown discoloration of vascular tissue accompanied by elevated levels of free amino acids and reducing sugars. Here we demonstrate that ZC disease induces selective protein catabolism in tubers through modulating protease inhibitor levels. Soluble protein content of tubers from CLso-infected plants was 33% lower than from non-infected plants and electrophoretic analyses revealed substantial reductions in major tuber proteins. Patatin (~40 kDa) and ser-, asp- (22 kDa) and cys-type (85 kDa) protease inhibitors were either absent or greatly reduced in ZC-afflicted tubers. In contrast to healthy (non-infected) tubers, the proteolytic activity in CLso infected tubers was high and the ability of extracts from infected tubers to inhibit trypsin (ser-type) and papain (cys-type) proteases greatly attenuated. Moreover, extracts from CLso-infected tubers rapidly catabolized proteins purified from healthy tubers (40 kDa patatin, 22 kDa protease inhibitors, 85 kDa potato multicystatin) when subjected to proteolysis individually. In contrast, crude extracts from non-infected tubers effectively inhibited the proteolytic activity from ZC-afflicted tubers. These results suggest that the altered protein profile of ZC afflicted tubers is largely due to loss of ser- and cys-type protease inhibitors. Further analysis revealed a novel PMSF-sensitive (ser) protease (ca. 80-120 kDa) in CLso infected tubers. PMSF abolished the proteolytic activities responsible for degrading patatin, the 22 kDa protease inhibitor(s) and potato multicystatin by CLso infected tubers. The disease-induced loss of patatin and protease inhibitors therefore appears to be modulated by ser-type protease(s). The selective catabolism of proteins in ZC-afflicted tubers undoubtedly affects downstream aspects of carbohydrate and amino acid metabolism, which is ultimately reflected by the accumulation of reducing sugars, free amino acids and reduced sprouting capacity.

FORMULA BERBAHAN AKTIF PSEUDOMONAD FLUORESEN DAN PENGARUHNYA TERHADAP PERKEMBANGAN PENYAKIT LAYU PADA CABAI

Formula with active ingridient of fluorescent pseudomonads and its influence on wilt disease development of pepper. The purpose of this study was to synergize organic fertilizer with biopesticides of fluorescent pseudomonads 122 to be the best formula to increase the resistance of pepper plants to wilt disease. The study used a completely randomized design (CRD), which consisted of four treatments. The treatments were forms of active ingredient formula of fluorescent pseudomonads (powder, pellet, granule, and liquid). The effectiveness of the formula in suppressing development of the disease was measured by observing the incubation period, index of the disease and discoloration of vascular tissue of pepper. The results showed that the disease incubation period of plant treated with liquid and powder formula was longer than formula with granule and pellet. Plants treated with powder formula showed the lowest disease index compared to control, liquid, pellet and granule formula. Disease index and discoloration in vascular tissue was also of the lowest value when the plants were treated with powder formula. This result indicate that active ingredient of fluorescent pseudomonads formulated in powder was the most effective to inhibit the development of wilt disease on pepper caused by Fusarium sp. and Ralstonia solanacearum.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Avocado Trees in Greece.

Avocado (Persea americana) is an important crop for Chania, Crete, Greece, and is grown on more than 800 ha. In November 2013, 4-year-old trees in a new avocado grove of cv. Hass grafted onto the rootstock 'Bacon,' previously planted in citrus trees, showed symptoms of yellowing, leaf fall, twig and branch dieback and vascular tissue discoloration. Disease incidence was estimated at 2.3% (12 out of 530 trees affected). A fungus was consistently and readily isolated from symptomatic vascular tissue, previously surface-disinfested with 95% ethanol, on acidified potato dextrose agar (APDA). After 7 days, slow-growing colonies were transferred to PDA and the growth rate of the fungus was 2.9 mm/day at 24°C in the dark. Microscopic observations revealed hyaline hyphae with many irregular, dark microsclerotia measuring 40 to 200 × 30 to 75 μm (average 94.5 × 50.3 μm) developing after 21 days of growth. Hyaline, elliptical, single-celled conidia measuring 2.8 to 7.5 × 2.5 to 4.3 μm (average 4.8 × 3.1 μm) developed on verticillate conidiophores. For molecular characterization, Verticillium dahliae specific primer pair ITS1-F/ITS2-R that amplifies the rRNA internal transcribed spacer (ITS) region was used (2). Band of expected size was amplified, sequenced, and deposited in GenBank (Accession No. KJ818294). On the basis of morphological characteristics (3) and a BLAST search with 100% identity to the published ITS sequence of a V. dahliae isolate in GenBank (KC834733.1), the fungus was identified as V. dahliae. Five 1-year-old avocado plants of cv. Hass, grafted onto the rootstock 'Bacon,' were used for pathogenicity tests. Artificial inoculation was performed by making a 5.0 × 3.5 mm hole in the rootstock trunk, injecting approximately 40 μl of a 2.8 × 107 conidia/ml suspension into the vessels (spores were introduced passively), sealing with Vaseline, and covering with adhesive paper tape. Five control plants were mock inoculated with sterilized distilled water. Disease symptoms that appeared 18 days post artificial inoculation were similar to those observed under natural infection conditions. Thirty-five days post artificial inoculation, disease incidence was 80%, whereas the percentage of positive V. dahliae re-isolations from infected tissues was 95% (96.7 and 93.3% from rootstock and graft, respectively). The extent of vascular tissue discoloration from the point of inoculation ranged from 11 to 62 cm, whereas V. dahliae was successfully re-isolated even from the end of the graft (approximately 60 cm above the initial inoculation point), thus confirming Koch's postulates. Neither symptoms nor positive isolations were observed in control plants. The pathogenicity test was repeated twice with similar results. Verticillium wilt of avocado has been observed in several countries including Argentina, Chile, Ecuador, Israel, Mexico, Morocco, Spain, and the United States (1). To the best of our knowledge, this is the first report of Verticillium wilt on avocado in Greece. This disease could potentially be an increasing problem in areas where young avocado trees are established on land previously planted in vegetable crops.