First Report of Lasiodiplodia pseudotheobromae Causing Collar Rot of Peanut in Shandong Province, China

In August 2019, a collar rot of peanut was observed in several fields in Qingdao, Shandong province, China. Disease survey was conducted in several peanut fields. Less than 5% plants exhibited various symptoms, including brown or black stem rot, pod rot, leaf chlorotic, wilted, and even dead. Symptomatic stems were cut into small pieces, surface disinfested with 70% ethanol for 1 min, 1% NaClO for 2 minutes, rinsed three times with sterile water, and dried on sterile filter papers. Pieces then were plated on potato dextrose agar (PDA) media and incubated at 25°C in darkness. Fungal cultures were initially white, then turned gray, and eventually turned black, and aerial hyphae were dense, fluffy. Conidia were ellipsoidal, initially hyaline, unicellular, 14.3 to 21.1 × 8.7 to 13.2 µm (n = 50), and mature conidia showed dark brown, with a central septum, and longitudinal stripes. Molecular identification was performed by sequencing ITS with ITS1/ITS4 (White et al., 1990) and beta tubulin gene with Bt2a/Bt2b (Glass and Donaldson, 1995) of a representative isolate ZHX9. ITS and beta tubulin regions (OK427342 and OK489788) of ZHX9 obtained 99.62 and 100% similar to L. pseudotheobromae (KF766193 and EU673111), respectively. Phylogenetic analysis was done using Neighbor-Joining (NJ) analysis based on those gene sequences. The microorganism we have isolated was identified as L. pseudotheobromae based on molecular analysis and morphological characteristics. For pathogenicity assay, twelve ten-days-old peanut (Zhonghua No.12) seedlings were each inoculated with one mycelial plug (8 mm in diameter) by placing the inoculum on the base of the stem. Twelve plants were each inoculated with a plug of non-colonized PDA as controls. Plants were incubated in a growth chamber (30°C in the day and 25°C at night, a 12-h photoperiod and 80% RH). Necrotic lesions were observed on stems of all inoculated seedlings 5 days after inoculation, whereas control plants remained asymptomatic, and L. peudotheobromae was consistently re-isolated from symptomatic stem. In Asia, peanut collar rot caused by L. teudotheobromae has been reported in India, Indonesia, North Vietnam (Nguyen, et al., 2006) and China (Guo, et al., 2014), but collar rot caused by L. pseudotheobromae has not been reported. To our knowledge, this is the first report of L. peudotheobromae causing collar rot on peanut in China. These results will provide crucial information for studying on epidemiology and management of this disease.

First Report of Sea buckthorn Stem Wilt Caused by Fusarium proliferatum in Liaoning, China

Sea buckthorn（Hippophae rhamnoides L.） is a flowering shrub native to cold-temperate regions of Eurasia, which is also valuable for its berries and leaves containing various vitamins and flavonoids (Pundir et al. 2021). In late June 2020, high mortality (more than 70%) was observed in sea buckthorn in a 1.6-ha seedling nursery in Chaoyang City, Liaoning province, China, where 16 Chinese and Russian cultivars (cv.) had been planted since 2014 (cv. Shenqiuhong, eshi01 through eshi15). The mortality of two introduced sea buckthorn varieties (eshi02, eshi04) was 100% (125 trees died in total). The symptoms include massive drooping leaves and dried-up stems on 6-year-old infected trees. Pieces of tree roots and stems with brown discoloration in the xylem vessels were selected. Small tissue fragments (0.2-0.5 cm) were surface disinfested (3 min in 75% ethanol, rinsed with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues, and a representative isolate (LC-1) was harvested. Genomic DNA was extracted for amplification and sequencing of the partial translation elongation factor-1α (EF1 and EF2 primers, accession Nos. MZ669853) (O’Donnell et al. 1998) and RNA polymerase II second largest subunit (RPB2) (7cf/11aR primers, accession Nos. MZ669854) (O’Donnell et al. 2007). The sequences were further analyzed at the Fusarium MLST (https://fusarium.mycobank.org/) for identity confirmation, and showed 99.8% (over 95.2% query coverage) and 96.4% (over 88.4% query coverage) similarity to Fusarium proliferatum (NRRL 13584, 13591). Isolates on Spezieller Nahrstoffarmer agar (SNA) produced abundant aerial white mycelia and yellow pigmentation. The 30 macroconidia measured ranged from 28.5 - 62.5 × 3.2 – 5.4 μm, were thin, slender, with 3-5 septa. The aseptate microconidia ranged from 4.7 – 13.6 × 2.2 – 4.3 μm (n = 30). Pathogenicity tests were performed on healthy, potted 1-year-old sea buckthorn seedlings (cv. eshi05) using two isolates in a greenhouse at 25 °C, 80% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedlings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedlings were inoculated with PDA plugs as controls. Six to ten days after inoculation, color of the leaves in the middle of the stems was variegated, and then dark necrotic lesions on leaf margins were observed. Three weeks after inoculation, 80% of inoculated stems were wilted, while control plants remained asymptomatic. The pathogen was consistently re-isolated and the recovered isolates were identified as F. proliferatum by amplifying the EF-1α gene. The typical symptoms on inoculated plants were dark to brown necrotic lesions on chlorotic leaves initially, and black withered stems in the terminal stage, similar to those observed on sea buckthorn trees infected with Fusarium sporotrichioides in Gansu and Heilongjiang provinces (Song et al. 2010; Xia et al. 2021). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. proliferatum in Liaoning province, China, which will be beneficial for expanding knowledge of Fusarium disease in sea buckthorn and provide more information for sustainable disease management in sea buckthorn.

Etiology of Botryosphaeria Panicle and Shoot Blight of Pistachio (Pistacia vera) caused by Botryosphaeriaceae in Italy

Pistachio (Pistacia vera) is an important crop in Italy, traditionally cultivated in Sicily (southern Italy) for several decades now. In recent years, new orchards have been planted in new areas of the island. Field surveys conducted in 2019 revealed the presence of symptomatic trees showing shoot dieback, cankers, fruit spots, and leaf lesions. Isolations from symptomatic samples consistently yielded fungal species in the Botryosphaeriaceae family. Identification of collected isolates was conducted using morphological and molecular analyses. Morphological characterization was based on conidia measurements of representative isolates and also effects of temperatures on mycelial growth was evaluated. DNA data derived from sequencing the ITS, tef1-α and tub2 gene regions were analyzed via phylogenetic analyses (Maximum Parsimony and Maximum Likelihood). Results of the analyses confirmed the identity of Botryosphaeria dothidea, Neofusicoccum hellenicum and Neofusicoccum mediterraneum. Pathogenicity tests were conducted on detached twigs and in the fields both on shoots as well as on fruit clusters using the mycelial plug technique. The inoculation experiments revealed that among the Botryosphaeriaceae species identified in this study N. hellenicum (occasionally detected) and N. mediterraneum were the most aggressive based on lesion length on shoots and fruits. N. mediterraneum was the most widespread among the orchards while B. dothidea can be considered a minor pathogen involved in this complex disease of pistachio. Moreover, to our knowledge, this is the first report of N. hellenicum in Italy.

First Report of Brown Rot Caused by Monilinia polystroma on Apple in Italy

Brown rot is a common apple disease in Italy, caused by Monilinia fructicola, M. laxa and M. fructigena (Martini et al. 2013). In September 2020, in a ‘Jeromine’ apple orchard under integrated pest management located in Scarnafigi (44°39'N, 7°33'E, north-western of Italy), fruits (8.6%) showing brown to blackish firm lesions (6.0 to 8.0 cm diameter) were observed. In some fruits, rots were covered by yellowish stromata. Two isolates (MPI1; MPI2) were obtained from two symptomatic apples and cultured on potato dextrose agar (PDA) for 7 days at 25°C in 12-h light/12-h dark regime. A white-to-greyish mycelium with slightly undulate margins and irregular, black stromata developed on PDA after 12 days incubation. Conidia, observed in branched monilioid chains, (Suppl. Fig. 1) were one-celled, globose, limoniform, hyaline, 38 to 58 μm (mean: 48) × 20 to 44 μm (mean: 33). Based on morphology, the isolates were tentatively identified as Monilinia polystroma (G.C.M. Leeuwen) Kohn. A polymerase chain reaction with primers ITS1 and ITS4 was performed on internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene. The sequenced amplicons (435 bp - 445 bp; GenBank Accession No. MW600854; MW600855) showed 100% identity to the reference isolate of M. polystroma (HQ846944) and to other isolates from apples (AM937114; JX315717) and plum (GU067539). The ITS region of M. polystroma had five nucleotides to distinguish it from the closest species M. fructigena (Zhu et al. 2016; MH862738) (Suppl. Fig. 2). The pathogenicity of both isolates was tested on mature ‘Jeromine’ apples (10.1% total soluble solids). Three replicates of six apples per isolate were surface disinfected with 1% NaClO. A mycelial plug (5 mm) from colony grown on PDA was inserted using a cork borer into a hole (6 mm) in each fruit (Vasić et al. 2016). Apples inoculated with sterile PDA plugs were used as control. Fruits were placed at 22 ± 1 °C, 85% relative humidity and 12 h light/12 h dark regime. Lesion size was measured after 3, 6 and 9 days of incubation. All inoculated fruits developed typical brown rot symptoms 6 days after inoculation and yellowish stromata appeared on the surface; control fruit remained healthy (Suppl. Fig. 3). The virulence of both isolates was statistically similar (Suppl. Table 1). M. polystroma was reisolated from all inoculated fruits and confirmed by molecular methods. This is the first report of M. polystroma on apple in Italy. M. polystroma was previously reported on apple in Hungary (Petróczy et al. 2009), on apricot in Switzerland (Hilber-Bodmer et al. 2012), on peach and pear in Italy (Martini et al. 2014; 2015), on plum in China (Zhu et al. 2016), and on apple in Serbia (Vasić et al. 2018). The emergence of this pathogen for pome and stone fruit production in Europe stimulates to study its biology and epidemiology, and its fitness and management, as compared to the other endemic Monilinia species.

The Toxin Cercosporin is a Virulence Factor for Infection of Coffee by Cercospora coffeicola

ABSTRACTBrown eye spot, caused by Cercospora coffeicola, causes significant losses in both quality and quantity of coffee production. As many Cercospora spp. produce the photoactivated toxin cercosporin, this study aimed to determine the role of cercosporin in C. coffeicola pathogenesis by creating disruption mutants unable to produce the toxin. Six C. coffeicola isolates from Brazilian fields, representing organic and conventional production systems in the Minas Gerais state, were evaluated for their ability to produce cercosporin in vitro. Toxin production varied among isolates, ranging from 3.5 – 25.3 µM/ 5 mm mycelial plug; production was undetectable in one isolate. The C. coffeicola homolog of the polyketide synthase gene (CTB1) involved in cercosporin production was amplified using a degenerate primer strategy. The 7044 nt ccCTB1 gene sequence was 90.3% identical to the cnCTB1 gene in Cercospora nicotianae and encoded a putative protein of 2196 amino acids with 98.2% similarity and 97.5% identity to its counterpart in C. nicotianae. Transformation of two isolates of C. coffeicola with a CTB1 disruption construct resulted in the recovery of six ctb1 disruption mutants. All of the ctb1 disruptants were deficient in cercosporin production. Disruption mutants did not differ significantly from the wild type for either growth or sporulation, but were significantly altered in virulence on coffee. As compared to wild type, time to lesion development was significantly increased and numbers of lesions were significantly decreased in coffee plants inoculated with ctb1 disruption mutants. These results show that cercosporin toxin is a virulence factor for C. coffeicola infection of coffee.

Phylogeny, Distribution, and Pathogenicity of Lasiodiplodia Species Associated With Cankers and Dieback Symptoms of Persian Lime in Mexico

Persian lime (Citrus latifolia Tan.) is an important and widely cultivated fruit crop in several regions of Mexico. In recent years, severe symptoms of gummosis, stem cankers, and dieback were detected in the Persian lime-producing region in the states of Veracruz and Puebla, Mexico. The aims of this study were to identify the species of Lasiodiplodia associated with these symptoms, determine the distribution of these species, and test their pathogenicity and virulence on Persian lime plants. In 2015, symptomatic samples were collected from 12 commercial Persian lime orchards, and 60 Lasiodiplodia isolates were obtained. Fungal identification of 32 representative isolates was performed using a phylogenetic analysis based on DNA sequence data of the internal transcribed spacer region and part of the translation elongation factor 1-α and β-tubulin genes. Sequence analyses were carried out using the Maximum Likelihood and Bayesian Inference methods. Six Lasiodiplodia species were identified as Lasiodiplodia pseudotheobromae, Lasiodiplodia theobromae, Lasiodiplodia brasiliense, Lasiodiplodia subglobosa, Lasiodiplodia citricola, and Lasiodiplodia iraniensis. All Lasiodiplodia species of this study are reported for the first time in association with Persian lime in Mexico and worldwide. L. pseudotheobromae (46.9% of isolates) was the most frequently isolated species followed by L. theobromae (28.1%) and L. brasiliense (12.5%). Pathogenicity on Persian lime young plants using a mycelial plug inoculation method showed that all identified Lasiodiplodia species were able to cause necrotic lesions and gummosis, but L. subglobosa, L. iraniensis, and L. pseudotheobromae were the most virulent.

Chitosan biostimulant controls infection of cucumber by Phytophthora capsici through suppression of asexual reproduction of the pathogen

The biopolymer chitosan is a derivative of chitin, which can promote plant growth and protect plants from phytopathogens. This study aimed to evaluate the efficacy of chitosan as a biostimulant and a biorational agent to protect cucumber plants from damping-off disease caused by <em>Phytophthora capsici</em>. Cucumber seeds were treated with a range of chitosan concentrations, viz. 0, 125, 250, and 500 ppm, to evaluate effect on seed germination and fresh root and shoot weight of the seedlings. Chitosan significantly (<em>p</em> ≤ 0.05) enhanced seed germination and root and shoot growth of cucumber in a dose-dependent manner up to 500 ppm. Application of in vitro chitosan suspension onto <em>P. capsici</em> mycelial plug suppressed growth of mycelia, formation of sporangia, and release of <em>P. capsici</em> zoospores at 125–500 ppm concentrations. Cucumber seedlings from chitosan-treated seeds showed enhanced resistance to damping-off disease caused by <em>P. capsici</em> compared to untreated control. Cucumber seedlings from 500 ppm chitosan seed treatment showed 100% disease resistance against damping off caused by <em>P. capsici</em>. These results suggest that chitosan could be used as a natural and environmentally safe alternative to a synthetic growth promoter and pesticide for sustainable production of cucumber.

Evaluation of Ornamental Tropical Plants for Resistance to White Mold Caused by Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a fungal pathogen that causes stem rot, crown rot, wilt, and death of many common annual flowering plants. Infested flower beds often suffer significant plant loss each year, and the identification of disease resistant plants would be a useful management tool. Caladium (Caladium ×hortulanum Birdsey), canna (Canna ×generalis L.H. Bailey), and elephant ear [Colocasia esculenta (L.) Schott] were evaluated for potential resistance to S. sclerotiorum. Plants grown in field conditions in Minnesota in 2012 and 2013 were inoculated through the application of sorghum grains colonized by S. sclerotiorum. The number of plants infected and percent of canopy dieback were recorded weekly for 3 months. The susceptibility of leaves, flowers, and below ground storage organs was also examined through direct inoculation of plant tissue with a mycelial plug of the pathogen in controlled environmental conditions favorable for disease development. Symptoms and progression of the infection were recorded after 24 days. Symptoms of infection on all three species were similar in field and controlled environments. Caladium plants were susceptible to S. sclerotiorum. Petioles, leaves, and corms developed a watery soft rot. Elephant ear was highly resistant to infection. Sclerotinia sclerotiorum infected only wounded or senescent tissue and did not result in significant symptoms under any conditions. Canna had partial resistance to the pathogen. Although canna petals were readily infected, infection of petioles was restricted to small necrotic lesions. Neither infection progressed to the main stem or resulted in plant death. This study indicates that canna and elephant ear have resistance to S. sclerotiorum and could be used in an integrated disease management program for infested landscape beds.

First Report of Phomopsis Stem Canker of Sunflower (Helianthus annuus) Caused by Diaporthe gulyae in Canada

During September 2012, Phomopsis stem canker was observed on sunflowers (Helianthus annuus L.) in a production field during seed filling with an average incidence of 15% in Morden, Manitoba (approximately 49°11′N and 98°09′W). The infected plants had elongated, brown-black lesions surrounding the leaf petiole, with numerous pycnidia, pith damage, and mid-stem lodging. Twenty sunflower plants were randomly sampled from the field. Isolations were made from the margins of the necrotic stems lesions by plating small pieces (5 mm) on potato dextrose agar (PDA) amended with 0.02% streptomycin sulfate. Plates were incubated at 25°C for 14 days under a 12-h photoperiod, and hyphal tips of white to grey colonies were transferred to PDA. Five isolates producing black pycnidia (occasionally with ostiolate beaks) and alpha conidia were tentatively identified as a Diaporthe sp. Alpha conidia were ellipsoidal, hyaline, and 6.5 to 8.5 × 2.5 to 3.5 μm. DNA was extracted from the mycelium of five isolates, and the ITS region was amplified and sequenced using primers ITS5 and ITS4 (4). BLASTn analysis of the 600-bp fragment (GenBank Accession Nos. KM391960 to KM391964) showed that the best match was Phomopsis sp. AJY-2011a strain T12505G (Diaporthe gulyae R.G. Shivas, S.M. Thompson & A.J. Young [3], Accession No. JF431299) from H. annuus with identities = 540/540 (100%) and gaps = 0/540 (0%). The five D. gulyae isolates were tested for pathogenicity on a sunflower confection inbred cv. HA 288 using the stem-wound method (2). Four-week-old sunflower plants (10 plants per isolate) were inoculated by wounding the stems on the second internode with a micropipette tip and placing a Diaporthe-infested mycelial plug on the wound. All plugs were attached to the wound with Parafilm. The pots were placed on the greenhouse benches at 25°C under a 16-h light/dark cycle. At 3 days after inoculation, dark brown lesions were observed on the stems extending upward from the inoculation site. Stem and leaves wilted, causing plant death 14 days after inoculation. Disease severity was calculated as a percentage of stem lesion (lesion length/stem length × 100%) at 14 days after inoculation. Significant differences (P ≤ 0.05) in disease severity were observed among D. gulyae isolates, which ranged from 34.9 to 100.0% (n = 5). Ten control plants similarly treated with sterile PDA plugs did not display symptoms. To complete Koch's postulates, D. gulyae was re-isolated from the inoculated stems, and the pathogen's identity was confirmed via sequencing of the ITS regions using primers ITS5 and ITS4 (4). The pathogen was not isolated from the control plants. D. gulyae was first reported as a pathogen on H. annuus in Australia and United States in 2011 (1,3). The pathogen was determined to be as or more aggressive than the other causal agents of Phomopsis stem canker (2,3), and its identification in both countries was circumstantially associated with increased incidence and yield loss in commercial production fields (1,3). In Canada, Phomopsis stem canker has been observed in sunflower fields over the last 10 years at low incidences, especially in years with above-normal temperatures during the sunflower growing season; however, the causal agent was not confirmed. To the best of our knowledge, this is the first report of D. gulyae causing Phomopsis stem canker on sunflowers in Canada. Since there is currently no known resistance to D. gulyae in sunflower, this newly discovered pathogen may become a threat to sunflower production in Canada. References: (1) F. Mathew et al. Phytopathology 101:S115, 2011. (2) F. Mathew et al. Phytopathology 103:S2.91, 2013. (3) S. M. Thompson et al. Persoonia 27:80, 2011. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Branch Rot of Lonicera japonica Caused by Sclerotium delphinii in China

Honeysuckle flower (Lonicera japonica Thunb.) is a perennial, traditional Chinese medicine plant, widely cultivated in China. In early June 2013, heavy branch rot infection was observed on L. japonica in an approximately 10,000-m2 field in Linyi, Shandong, China. The disease incidence was 30 to 40%. Early symptoms appeared as small, elliptoid, pale brown lesions on the branches. Lesions expanded into 50 to 100 mm long and 3 to 7 mm wide, brown, elongated spots. The upper branches wilted after the lesions expanded around the stems. A fungus was consistently isolated from stem lesions on potato dextrose agar (PDA) that was morphologically similar to S. delphinii, with white mycelium, round to irregularly shaped reddish-brown sclerotia that were 2 to 4 mm diameter (2). The identity of the fungus was confirmed by DNA sequencing of the internal transcribed spacer (ITS) region (GenBank Accession No. KJ145328), which was 99% homologous to those of other S. delphinii isolates (JN241578 and AB075314) (1). Pathogenicity tests were conducted with three 2-year-old seedlings grown in 20-cm-diameter pots at 25 to 30°C during experiments in greenhouse. Ten branches from the three plants pricked by needle were inoculated with a mycelial plug (0.4 cm diameter) harvested from the periphery of a 4-day-old colony. An equal number of branches pricked by needle serving as controls were mock-inoculated with plugs of PDA medium. Inoculated branches were covered with plastic bags for 24 h to maintain high relative humidity and incubated at about 25°C. Plugs were removed 48 h after inoculation. After 3 days, nine inoculated branches showed symptoms identical to those observed in the field under natural conditions, whereas controls remained symptom-free. Re-isolation of the fungus from lesions on inoculated branches confirmed that the causal agent was S. delphinii. Pathogenicity tests were repeated three times by the same methods with the same results. To our knowledge, this is the first report of S. delphinii infecting Lonicera japonica in China. References: (1) I. Okabe and N. Matsumoto. Mycol. Res. 107:164, 2003. (2) Z. K. Punja and A. Damiani. Mycologia. 88:694, 1996.