First Report of Fusarium avenaceum Causing Canker Disease on Kiwi Tree in China

In May 2021, canker symptoms were detected on ‘Xuxiang’ kiwi trees in southwestern Shaanxi (Hanzhong municipality; 107.27° E, 33.23° N) in China. Seven-year-old trees exhibited black necrotic lesions and cracked areas in the trunk (Figure 1). The symptoms were observed in approximately 10% of the trees in 6 orchards (31 ha in total). Application of commercial fungicides did not control the advancement of the pathogen, and infected trees were removed to control the spread. Three samples, approximately 1 cm2 in size, of symptomatic tissue were collected and surface sterilized in 2% NaOCl for 1 min, and washed with sterile ddH2O. Four isolates showing white mycelium with yellow pigmentation were obtained after 4 days of incubation on PDA, containing chloramphenicol (50 µg/mL), at 28 ºC. The pathogen was isolated from all collected samples. ITS, EF1-α, TUB2, RPB1 and RPB2 genes were amplified using ITS1/ITS4, EF1-728F/EF1-986R, T1/T22, RPB1-5F/RPB1-8R and RPB2-5F/RPB2-7cR (strain NJC06), or RPB2-c7F/RPB2-11aR (strains NJC07 and NJC08), primers, respectively. Two isolates shared the same sequences (strain NJC08). Obtained sequences were submitted to GenBank under accession numbers MZ669205 and OL347898-OL347899 (ITS), OL439731-OL439733 (EF1-α), OL439734-OL439736 (TUB2), OL439737-OL439739 (RPB1), and OL439740-OL439742 (RPB2). The sequences shared >99% (ITS; F. avenaceum CBS 128538, MH864972), >99% (EF1-α; F. avenaceum 55-2, MN473124), 100% (TUB2; F. avenaceum SICAUCC 18-0001, MK253102), >98% (RPB1; F. avenaceum NRRL 26911, MG282372), and >98% (RPB2; F. avenaceum SICAUCC 18-0001, MK396098; or F. avenaceum FRC R-09495, CQ915486) homology to multiple F. avenaceum strains. Molecular phylogenetic tree (Figure 2) was constructed using MEGA7 with Fusarium strains found causing rot in various hosts (Wang et al. 2015), and other fungal species, such as Cadophora nalorum, Diaporthe ambigua, D. australafricana, and Neofusicoccum parvum, which were reported to cause cordon dieback on kiwi tree in Chile (Diaz et al. 2021). Microscope observations after cultivation of all isolates on barley-honey-tryptone medium (Song et al. 2020) showed the presence of septate mycelium, fusiform microconidia (8-15 µm in length, containing between 0 and 3 septa; n = 77) and chlamydospores (n = 21), and agree with the morphology of F. avenaceum (Zhao et al. 2020). To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the trunk of 3-months-old ‘Xuxiang’ kiwi trees. Solutions containing 1 × 106 spores/mL (20 µL) of the isolates were injected in the wounds. Sterile ddH2O was used for the control experiment. Inoculated plants were maintained in a growth chamber at 28 °C and 80% relative humidity for 4 days. The pathogen was recovered from the canker lesions, which were similar to those observed in the orchards, and its identity was confirmed by sequence analysis. The pathogen only infected wounded trees, and probably invaded the orchards during the pruning in February 2021. F. avenaceum was reported to cause canker on almond tree (Stack et al. 2020), stem rot on Anthoxanthum aristatum and Polygonatum cyrtonema (Pieczul et al. 2018; Xu et al. 2019), and root rot on carrot, Coptis chinensis and wheat (Le Moullec-Rieu et al. 2020; Mei et al. 2020; Ozer et al. 2020). Recently, F. avenaceum was found causing fruit blotch in kiwi fruit in Anhui (China) (Zhao et al. 2020). Here, F. avenaceum was found causing canker disease in kiwi tree, demonstrating the host and tissue promiscuity of this pathogen. Kiwi is an important crop in China with nearly 1.5 million tons produced in 2019. This report will help to better understand the pathogens reducing kiwi production in China.

Morphology and molecular study of three new Cordycipitoid fungi and its related species collected from Jilin Province, northeast China

Cordyceps species are notable medicinal fungi in China, which are pathogenic on insects and exhibit high biodiversity in tropical and subtropical regions. Recently, three new Cordyceps species, Cordyceps changchunensis and Cordyceps jingyuetanensis growing on pupae of Lepidoptera and Cordyceps changbaiensis growing on larvae of Lepidoptera, were found in Jilin Province, China and are described, based on morphological and ecological characteristics. These three new species are similar to the Cordyceps militaris group, but are distinctly distinguishable from the known species. Cordyceps changchunensis, characterised by its small and light yellow to orange stromata which is occasionally forked, covered with white mycelium at the base of stipe, globose to ovoid perithecia, is macroscopically similar to Cordyceps militaris. Cordyceps changbaiensis is clearly discriminated from other Cordyceps species by its white to orange and branched stromata, clavate to cylindrical fertile apical portion, immersed and globose to ovoid perithecia. Moreover, unbranched, clavate and orange to light red stromata, almond-shaped to ovoid and immersed perithecia separate Cordyceps jingyuetanensis from other Cordyceps species. nrITS, nrLSU and EF-1α sequences were undertaken and phylogenetic trees, based on Maximum Likelihood and Bayesian Inference analysis showed that the three new species clustered with Cordyceps militaris, but formed individual clades, as well as confirmed the results of our morphological study.

Identification of the Causal Agent of Aqueous Spot Disease of Sweet Cherries (Prunus avium L.) from the Jerte Valley (Cáceres, Spain)

The pre and postharvest disease named ‘aqueous spot’ is an emerging risk for sweet cherries growing in Jerte Valley (Cáceres, Spain). Early stages of the disease appear in the tree, but it is usually detected after harvesting, during the postharvest period. Symptoms include the appearance of skin discolouration and translucency in the shoulder areas. At the most advanced stages, a mycelium of white colour partially or completely covers the fruit. This manuscript provides a detailed description of the microbes involved in this disease, such as bacteria, yeasts, and moulds. Microbes of different cherry cultivars were studied during two consecutive seasons (2019 and 2020). The counts of bacteria and yeast in damaged tissues were higher (7.05 and 6.38 log10 CFU/g for total aerobic mesophilic microbes and yeasts, respectively) than sound tissues (6.08 and 5.19 log10 CFU/g, respectively). The Enterobacterales order dominated the bacteria population. Among yeasts, Yarrowia lipolytica, in 2019, and Metschnikowia pulcherrima and Metschnikowia viticola, in 2020, were consistently isolated from all samples. The presence of moulds was inconsistently detected at the early stage of this disease by plate counts. However, microscopic observations revealed the presence of hyphae in cherry flesh. Different pathogenic moulds were identified, although white mycelium, identified as Botrytis cinerea by molecular methods, was consistently isolated at later stages. Inoculation tests confirmed the involvement of white-mycelium B. cinerea in the development of this new postharvest disease in the Jerte Valley. Its combination with Enterobacterales enhanced the evolution of rotting, whereas the combination with yeasts decreased and delayed the symptoms. This work presents the first report of a consortia of microorganisms implicated in the development of ‘aqueous spot’, an emerging disease in sweet cherry cultivars in the Jerte Valley.

First Report of Fusarium equiseti, Causing Fruit Rot Disease of Watermelon in Malaysia

Watermelon (Citrullus lanatus) accounts for almost 13% of all tropical fresh fruit production in Malaysia. They are grown, mostly in Johor, Kedah, Kelantan, Pahang, and Terengganu areas of Malaysia on 10,406 ha and yielding 172,722 Mt. In 2019, a new fruit rot disease was observed in two major production areas in Peninsular Malaysia. Disease symptoms included water-soaked brown lesions on the fruit surface in contact with the soil. The lesions enlarged gradually and ultimately covered the whole fruit with white mycelium leading to internal fruit decay. Disease surveys were conducted in December 2019 and November 2020 in fields at Kuantan, Pahang and Serdang, Selangor. Disease incidence was 10% in 2019 and 15% in 2020. Infected fruits were collected and washed under running tap water to wash off adhering soil and debris. Fruit tissue sections 1 to 2 cm in length were surface sanitized with 0.6% sodium hypochlorite (NaOCl) for 3 min. and washed twice with sterile distilled water. The disinfected air-dried tissues were then transferred onto potato dextrose agar (PDA) media and incubated at 25±2℃ for 3 days. Fungal colonies with whitish mycelium and pink pigment isolated using single spore culture. The pure cultures were placed onto carnation leaf agar (CLA), and the culture plates were incubated at 25±2℃ for 15 days for morphological characterization. On CLA, macroconidia were produced from monophialides on branched conidiophores in orange sporodochia. Macroconindia were thick-walled, strong dorsiventral curvature, 5 to 7 septate with a tapered whip-liked pointed apical cell and characteristic foot-shaped basal cell, 21.9 to 50.98 μm long and 2.3 to 3.60 μm wide. Typical verrucose thick chlamydospores with rough walls were profuse in chains or clumps, sub-globose or ellipsoidal. Based on morphological characteristics they were identified as Fusarium equiseti (Leslie and Summerell 2006). Molecular identification of both U4-1 and N9-1 pure culture isolates were carried out using two primer pair sets; internal transcribed spacer (ITS) ITS-1/ ITS-4 and translation elongation factor 1 alpha (TEF1-α) (EF-1/EF-2). A Blastn analysis of the ITS gene sequence of U4-1(MW362286) and N9-1 (MW362287) showed >99% similarity index to the reference gene sequence of F. equiseti isolate 19MSr-B3-4 (LC514690). The TEF1-α sequences of U4-1 (accession no. MW839563) and N9-1 (accession no. MW839564) showed 100% identity; with an e-value of zero, to the reference gene sequence of F. equiseti isolate URM: 7561 (accession no. LS398490). Each isolate also had a >99% identity with isolate NRRL 34070 (accession no. GQ505642) in Fusarium MLST database that belongs to the F. incarnatum-equiseti species complex (O’Donnell et al. 2015). Based on phylogenetic analysis of the aligned sequences (TEF1-α) by the maximum likelihood method, the U4-1 and N9-1 isolates were confirmed to be F. equiseti as was reported in Georgia, USA (Li and Ji 2015) and in Harbin, Heilongjiang Province, China (Li et al. 2018). Finally, the two pure culture isolates of U4-1 and N9-1 were used to fulfill Koch's postulates. Stab inoculations of five healthy watermelon fruits (cv. 345-F1 hybrid seedless round watermelon) were performed with a microconidial suspension of individual isolates (4x106 spores/mL). Five control fruits were stabbed with double distilled water. The inoculated fruits were incubated under 95% relative humidity at a temperature of 25±2℃ for 48 h followed by additional incubation inside an incubator at 25±2℃ for 8 days. Ten days post-inoculation, the control fruits showed no disease symptoms. However, inoculated fruits exhibited typical symptoms of fruit rot disease like water-soaked brown lesions, white mycelium on the fruit surface and internal fruit decay, which is similar to the farmer’s field infected fruits. The suspected pathogen was successfully re-isolated from the symptomatic portion of inoculated fruit and morphologically identified for verification. To our knowledge, this is the first report of F. equiseti causing fruit rot of watermelon in Malaysia. Malaysia exports watermelon year-round to many countries around the world. The outbreak of this new fruit rot disease could potentially pose a concern to watermelon cultivation in Malaysia.

Incidence, yield losses and symptomatology of sclerotinia stem rot (SSR) of Indian mustard (Brassica juncea L.) incited by Sclerotinia sclerotiorum

SSR hamper successful cultivation and causes significant yield losses globally including India. The SSR disease incidence ranged from 3.59 to 18.50 per cent in mustard growing areas of Rajasthan and Bharatpur district having the highest (18.50%) disease incidence. However, the Udaipur district had the lowest (3.59%) disease outbreak. SSR was responsible to cause 18.80 per cent overall yield losses in two consecutive years (Rabi 2016-17 and 2017-18). Highest yield loss (43.82%) was observed from Bharatpur district while minimum yield loss (3.51%) had recorded from Udaipur district. Characteristics symptoms, formation of white mycelium on host stem and development of sclerotia on/inside the stem, of SSR on mustard crop were noted during survey.

Morphology and molecular study of three new Cordycipitoid fungi and its related species collected from Jilin Province, Northeast China

Cordyceps are notable medicinal fungi in China, which pathogenic on insects and with high biodiversity in tropical and subtropical regions. Recently, three new Cordyceps species, Cordyceps changchunensis and Cordyceps jingyuetanensis grow on pupae of Lepidoptera, and Cordyceps changbaiensis grows on larvae of Lepidoptera, and one new record of Northeast China, Cordyceps taishanensis, are found in Jilin Province, China and detailed descriptions were obtained based on morphological and ecological characteristics. These three new species are similar to Cordyceps militaris group but distinctly distinguishable from known species. Cordyceps changchunensis, characterised by its small and light yellow to orange stromata that forks-like occasionally, covered with white mycelium at base of stipe, globose to ovoid perithecia, is macroscopically similar to Cordyceps militaris. Cordyceps changbaiensis clearly discriminated from other Cordyceps species by its white to orange and branched stromata, clavate to cylindrical fertile apical portion, immersed and globose to ovoid perithecia. Moreover, unbranched, clavate and orange to light red stromata, almond-shaped to ovoid and immersed perithecia differ Cordyceps jingyuetanensis from other Cordyceps species. ITS and nrLSU sequences are undertaken, and phylogenetic trees based on Maximum Likelihood analysis and Bayesian inference analysis confirmed the results of our morphological study. The phylogenetic result showed that the three new species gather with Cordyceps militaris but formed clades alone. Further analysis shows that the group of Cordyceps militaris mainly host on larvae of Lepidoptera.

First report of Erysiphe corylacearum, agent of powdery mildew, on hazelnut (Corylus avellana) in Romania

Romania has an area dedicated to hazelnut (Corylus avellana L.), covering 890 hectares as of 2019. During October 2020, powdery mildew symptoms were observed on the upper side of leaves of hazelnut ‘Tonda di Giffoni’ in two commercial orchards in Dudeștii Vechi, Romania (Fig. 1). The disease was present on 70% of the trees in planting, with at least 5 leaves per tree having powdery mildew. Micromorphological examination revealed amphigenous, hyaline, branched, septate mycelial patches of 2.3 to 3.6 μm in diameter. Conidiophores measured 24-60 × 5-6 (average: 45 × 6) μm and consisted of erect, cylindrical to flexuous foot cells, followed by 1-2 shorter cells. Ellipsoid, ovoid to doliform conidia were produced singly and they measured 19-35 × 16-24 (average: 28 × 19) μm. Chasmothecia were spherical, 75 to 107 (average: 88) μm in diameter. Nine to thirteen straight, sometimes flexuous, appendages measured 54 to 92 (average: 66) μm in length and they had five times dichotomous branched apices with curved tips (Fig. 2). Each chasmothecium contained three to five ellipsoid, ovoid to subglobose asci measuring 41-58 × 29-55 μm (average 52 × 43) μm. The asci contained four to eight ascospores measuring 13-24 × 11-15 (average 18 × 14) μm. Morphological identification was confirmed by sequencing the ITS-region of rDNA using two isolates from leaves, stored as frozen mycelium at -20°C. PCR was performed with Erysiphales-specific primer pair PMITS1/PMITS2 (Cunnington et al. 2003). The obtained sequences were deposited in GenBank (Accession n° MW423075, MW423076). Blast analysis of both sequences had 100% identity to ITS rDNA sequences of Erysiphe corylacearum from Azerbaijan (Abasova et al. 2018; Accession n° LC270863), Turkey (Sezer et al. 2017; KY082910), Switzerland (Beenken et al. 2020; MN82272), Iran (Arzanlou et al. 2018; MH047243), Italy (Mezzalama et al. 2020; MW045425) and 99% identity from Georgia (Meparishvili et al. 2019; MK157199). The sequences had a lower percent identity (83%) to Phyllactinia guttata (Accession n° AB080558) (Fig. 3). Pathogenicity was verified on one-year-old plants of C. avellana ‘Tonda di Giffoni’, which were artificially inoculated with a conidial suspension from infected leaves (n = 25). Inoculated plants were incubated at 20 to 28°C with 70 to 80% relative humidity. White mycelium appeared on the upper surface of the leaves at 8 to 10 days after inoculation. No symptoms were found on control plants sprayed with sterile water. The fungus present on inoculated leaves was morphologically identical to the original isolates from diseased trees from the field. E. corylacearum is native to East Asia and was previously reported in Japan on wild species of Corylus (Takamatsu et al. 2015; Accession n° LC009928). The pathogen most likely spread into Europe from east to west of Europe (Heluta et al. 2019), through the Caucasus, starting from Turkey, Azerbaijan, Georgia, and Iran. P. guttata was considered the only causal agent of powdery mildew on hazelnut in most countries, including Romania (Brown 1995). Compared to P. guttata, which generally develops a mycelium on the underside of leaves, E. corylacearum grows with a white mycelium on the upper side of the leaves. Recently, E. corylacearum on C. avellana was reported also in Ukraine (Heluta et al. 2019), from which it could have moved to Romania. Crop protection strategies for hazelnut should be revised according to the new pathogen occurrence.

First report of Colletotrichum siamense causing anthracnose on Erythrina crista-galli in China

Erythrina crista-galli L. (Fabaceae) is a popular ornamental plant in tropical and subtropical regions of South Asia. In October 2019, anthracnose-like lesions were observed on the leaves of E. crista-galli planted in Haikou, China. 5-30% of leaves were infected. At first, the circular spots of 1-2 mm in diameter were reddish-brown on the leaves, and then enlarged to circular, subcircular or irregular spots with reddish-brown center and surrounded by a diffuse yellow margin. Neighboring spots sometimes coalesced. Under continuously wet or humid conditions, the lesions expanded quickly, and became gray, subcircular or irregular spots covered by grayish-white mycelium and orange-pink conidial masses. Diseased leaves eventually fell off the trees. To identify the pathogen, diseased leaves were sampled from four gardens. Leaf tissues (5×5 mm) were cut from the margins of typical symptomatic lesions, surface-sterilized in 1% sodium hypochlorite for 1 min, plated on potato dextrose agar (PDA) medium, and incubated at 28.0±0.5℃ in the dark. Similar fungal colonies were obtained from all plated tissues after 3 days. The single-conidium colonies of all isolates were white to pale gray and cottony with visible orange conidial masses. Conidia were one-celled, aseptate, hyaline, straight, cylindrical to fusiform with obtuse ends, and ranged from 14.2-18.6 µm (16.4 µm)× 3.8-5.4 µm (4.7 µm) (n=100). After germination, conidia formed single, brown, oval or slightly irregular appressoria ranging from 8.0 to 11.8 μm (9.6 µm), and from 4.8 to 6.0 μm (5.4 µm). Sexual stage was absent. These characteristics of conidia and appressoria were matched with C. siamense belonging to the C. gloeosporioides complex (Prihastuti et al. 2009; Yang et al. 2009; Weir et al. 20012; Hu et al. 2015). To accurately identify the species, DNA was extracted from four purified isolates (JG-1, JG-3-1, SWS-1-3, SWS-2-1) （Fu et al. 2019）. The internal transcribed spacer of rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT) and chitin synthase (CHS) genes were amplified and sequenced. The nucleotide sequences were all deposited in GenBank (ITS: MT229427-MT229430, GAPDH: MT250821-MT250824, CAL: MT258893-MT258896, ACT: MT258897-MT258900 and CHS: MT258901-MT258904). Multi-locus phylogenetic analyses (ITS, GAPDH, CAL, ACT and CHS) (Weir et al. 2012) showed that the four isolates were clustered with C. siamense, which was in accordance with BLAST results. Pathogenicity tests of the four isolates were repeated three times on detached leaves (Ji et al. 2019). The conidial suspension (1×106 conidia/mL) was prepared using the conidia from 10-day-old cultures grown on PDA. Two 20-µL drops of conidial suspension were inoculated on non-wounded young healthy leaves, and each isolate was inoculated on 10 leaves. Two 20-µL drops of sterile water were inoculated on non-wounded young healthy leaves as control. The samples were maintained in containers at a relative humidity of 90± 5 per cent inside and 28℃ with a 12-h photoperiod. Gray, subcircular spots similar to the field disease symptoms were observed on the all inoculated leaves after 7 days, whereas no visible symptoms appeared on the non-inoculated leaves. The pathogen was re-isolated from inoculated leaves thus fulfilling Koch’s postulates. C. gloeosporioides has been previously reported as a pathogen causing leaf spot on Erythrina (E. indica var. picta, E. variegata var. orientalis) in Guam in 1983 and Brazil in 2012. (Russo et al. 1983; Oliveira et al. 2012). To our knowledge, this is the first report of C. siamense causing leaf spot of E. crista-galli in China.

Botrytis prunorum Associated to Vitis vinifera Blossom Blight in Chile

Table grapes are highly susceptible to Botrytis cinerea infections during the bloom period. After reaching the flower development stage, B. cinerea remains quiescent until berry ripening or gives rise to blossom blight under specific climate conditions. A research study was conducted on the Chilean Central Valley during the 2018–2019 growing season. Flowers of Vitis vinifera cv. Thompson Seedless were collected and B. cinerea was isolated together to a second and morphologically different species, characterized by white mycelium and low to no sporulation (11.4% of total isolates). Three randomly selected isolates within this population were genetically examined and identified as Botrytis prunorum based on a phylogenetic multilocus approach using partial regions of genes RPB2, HSP60, and G3PDH or NEP1 and NEP2. Pathogenicity tests showed that B. prunorum infects and causes wilting in healthy table grape flowers. B. prunorum isolates were able to infect Thompson Seedless berries, inducing lesions between 13.11 and 41.53% with respect to the lesion diameter generated by B. cinerea B05.10. The fungicide sensitivity was evaluated. The three genetically characterized isolates were sensitive to boscalid and to cyprodinil/fludioxonil mixture with a mean EC50 value of 5.5 µg/ml and 0.065 µg/ml, respectively. However, loss of sensitivity to fenhexamid was determined, with a mean EC50 value of 5.13 µg/ml. Our understanding about blossom blight in V. vinifera has been limited to B. cinerea. Here we associated B. prunorum as a second causal agent of this disease in Chile. This data represents a first approach to the epidemiological characteristics of B. prunorum associated with blossom blight in table grapes.

Sclerotium rolfsii, Penyebab Penyakit Busuk Pangkal Batang pada Hippeastrum sp.

Sclerotium rolfsii, a the Causal Agent of Stem Rot Disease on Hippeastrum sp.Symptoms of stem rot that cause Hippeastrum sp. or red lily wither, leaves turn yellow, and eventually die found at Mangliawan Village, District of Pakis, Malang - East Java. The purpose of this study was to identify the pathogens that cause root rot disease on lily plants and find out their host range. Sclerotium from the symptomatic base of the plant was isolated on potato dextrose agar medium. Fungus was identified based on the morphological characteristics of the colonies and mycelium. Host range test of pathogen was carried out by manual inoculation on Rain lily (Zephyranthes) St. Bernard's lily (Chlorophytum) and Beach Spider lily (Hymenocallis). The results of the identification showed that the fungus had white mycelium and formed sclerotium. Sclerotium is irregularly rounded, white when young, and dark brown when ripe, and forms 10 days after incubation. In hyphae, there are branching, septa, and clam connections. Based on the morphological characteristics of the disease the fungus was identified as Sclerotium rolfsii. In the host range test, the fungus was able to infect rain lilies and paris lilies, but not in spider lilies. This is the first report of S. rolfsii infection in lily in Indonesia.