First report of Stemphylium eturmiunum causing leaf spot and black spot on apple in Zhaotong, Yunnan, China

Apple is the largest fruit tree crop in the world, and China is the largest apple-producing County in the world. Zhaotong, Yunnan Province is a typical cold and mountainous apple-producing area in China. However, apple production is threatened by diseases during the entire growing season, and among them, apple leaf spot and fruit black spot are severe. In previous reports, the main pathogen causing apple leaf spot and fruit black spot was Alternaria sp. (Lior, et al, 2017), while different pathogens were identified. In the current study, seven red Fuji apple fruit with typical black spot samples were collected randomly in Dongda company orchard, Sujiayuan town, Zhaotong, Yunnan on March 25, 2021. The spots on the surface of these apples appear rounded, the diseased parts turn brown or black in colour and the flesh became soften and rotten. The tissues of fruit epidermis at the edge between diseased and healthy parts were cut, soaked in 75% alcohol for 30 s, washed with sterile water three times, and air-dried. Five pieces of tissue were placed on PDA medium amended with rifampicin (50 mg/ml) and incubated in the dark at 25 ℃ for 3-5 days. After colonies grew, mycelial clumps were picked out from the edges of the colonies, transferred to new PDA plates, and incubated at 25 ℃ for 6 days. The diameter of the colonies reached up to 5.7 cm. A representative isolate was retained for further work and was named P6-3-1. The hyphae were white and dense at an early stage, the culture medium on the underside became yellow and the middle parts of the colonies were darker. With maturity, hyphae were clumped, became red with other colors interspersed, and the medium became dark red. Light brown spores were produced, with more vertical septa and fewer transverse septa. Two to three transverse septa were generally observed with obvious constriction at the transverse septa. Average spore size was 22.83 µm ± 2.04 µm × 14.58 µm ± 1.97 µm. DNA was extracted from mycelium, purified and amplified with two pairs of primers, ITS1/ITS4 (White et al. 1990) and gpdF/gpdR (Marcos P. S. Câmara, et al. 2002). The PCR products were sequenced and deposited in GenBank (accession NO.OK560128 and OK627661 ). The similarity of ITS sequences between the isolate and MH843733 (Stemphylium eturmiunum strain ST14) was 100%, and that of gpd sequences between the isolate and MH843728 (Stemphylium eturmiunum strain ST20) was 100%. The maximum parsimony method of Mega7.0 was used and demonstrated that the studied isolate converged to the same branch as Stemphylium eturmiunum. Koch's postulates was applied to identify the pathogenicity of this isolate. A disc of P6-3-1-culture on PDA (5 mm in diameter) was placed on apple leaves and fruit wounds. Sterile PDA was used as a control. All plants were kept in a growth chamber at 25-30 ℃. Four days after inoculation, the disease spot was observed on the inoculated sites and fruit, and with the extension of incubation time, the diseased spots continue to grow, and the leaf spots were not limited by the veins. The pathogen was re-isolated from the inoculated leaves and fruit, satisfying Koch’s postulates. This pathogen can also cause postharvest rot of sweet cherry (Alice Spadoni, et al, 2020), postharvest rot on tomato (Prencipe Simona, et al, 2021), etc. This is the first report that Stemphylium eturmiunum can cause apple leaf spot and fruit black spot in Yunnan province, China. The apple black spot caused by Stemphylium eturmiunum was accurately identified. By distinguishing between the two similar diseases mentioned above, resistance to the host and management practices can be accrued based on the characteristics of the pathogen, its epidemiological pattern and the choice of an effective chemical fungicide.

First Report of Brown Leaf Spot of Rice (Oryza sativa L.) Caused by Bipolaris sorokiniana in Pakistan

Brown leaf spot of rice is one of the major seed-borne diseases and can diminish grain production up to 52% (Barnwal et al. 2013). In 2018, infected leaf samples showing the typical symptoms of brown spots were collected from the vicinity of the University of Agriculture, Faisalabad (31°26'10.3"N 73°03'35.1"E). The symptoms were brown-dark spots, with gray-light gray or brown centers surrounded by dark margins and with chlorotic halos and of oval or cylindrical shapes (5 to 9 mm in diameter). Disease incidence averaged 61% across the seven fields observed. Leaves were collected from the seven infected fields and symptomatic leaf tissues of 5 mm2 were excised from representative necrotic spots in each. These tissues were surface disinfected with 70% ethanol, rinsed with sterile distilled water (SDW), dried by blotting on paper, and placed on potato dextrose agar medium. For pathogen growth, the plates were placed at 25oC (±2oC) with a 12-hour photoperiod for 5 days. Five samples from each of the infected fields were taken for pathogen isolation and among them ten isolates were sub-cultured and purified by using the single spore method. The resulting fungal colonies were fluffy and ranged in color from grayish black/black to light brown. Fifteen conidia were measured that are olivaceous-brown to dark brown in color, elliptical to oblong with narrow (tapered) ends, with 3-10 septa and 35.6-65.4 µm in length x 13.1-25.7 µm in width. Conidiophores were yellowish-brown, geniculate, and solitary (Pratt 2003). For molecular studies, rDNA of the internal transcribed spacer (ITS) region, translation elongation factor (tef), RNA polymerase II second largest subunit (rpb2) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified by using the primers ITS1F/ITS4R (White et al. 1990), EF1-983F/EF1-2218R (Rehner and Buckley 2005), 5F2/7CR (O’Donnell et al. 2007), and GPD1/GPD2 (Berbee et al. 1999) respectively. The sequence of all the amplified gene regions of one SUL-1 isolate was deposited into GenBank with accession numbers MN314844 (ITS), MN326866 (tef), MN990457 (rpb2) and MN990456 (gpd). BLASTn queries of the obtained sequences (ITS, tef, rpb2 and gpd) showed 99-100% homology with the corresponding nucleotide sequences of B. sorokiniana (GenBank accession nos. GU480767, MF490855, LT715652 and MK558818 respectively). To fulfill the Koch’s postulates, twenty rice plants (cv. Basmati-385) were sprayed at 2 to 3 leaf stages by using the two representative isolates with a spore suspension of 105 spores/ml. SDW was sprayed on ten control plants. The plants were covered with polyethylene bags to keep the moisture contents and incubated at 25oC (±2oC) for 7 days. After a week, same symptoms as those described above were observed. In the repeated experiment, B. sorokiniana was re-isolated from the infected rice leaves and confirmed morphologically; fulfill the Koch’s postulates. With grave worry, the other species of the genus Bipolaris (B. oryzae, and B. victoriae) have also been found to the cause brown leaf spot of rice (Motlagh and Kaviani 2008). To our knowledge, this is the first report of Bipolaris sorokiniana causing brown leaf spot of rice in Pakistan. Because rice is highly consumable grain in Pakistan, so the rapid spread of this disease in the rice farming areas is of a serious concern.

First Report of a Leaf Spot Disease of Tobacco Caused by Pseudomonas cichorii in China

Tobacco (Nicotiana tabacum L.), one of the chief commercial crops, is wildly cultivated worldwide. In June 2020 and 2021, an unknown bacterial leaf spot on tobacco was found in Hezhou and Hechi City, Guangxi, China. 30% of the tobacco were affected and the rate of diseased leaves reached about 10% in the field under high temperature and rainstorm. The disease mainly damaged the middle and top leaves of tobacco plants at vigorous growing stage. The initial symptoms were water-soaked spots on the frontal half of a leaf, and then expanded into circular to irregular spots with a yellow halo at the edge. The spots mostly appeared dark brown at high air humidity, while yellow brown at low humidity and exhibited a concentric pattern. In severe cases, the lesions coalesced and the whole leaf was densely covered with lesions, resulting in the loss of baking value. A bacterium was consistently isolated from diseased leaf tissues on nutrient agar (NA). Growth on NA was predominantly grayish white circular bacterial colonies with smooth margins, and the bacterium is rod-shaped, gram-negative and fluorescent on King’s B medium. Seven isolates (ND04A-ND04C and ZSXF02-ZSXF05) were selected for molecular identification and pathogenicity tests. Genomic DNA of the bacterium was extracted and the housekeeping gene of cts (encoding citrate synthase) was amplified with the primers cts-Fs/cts-Rs (forward primer cts-Fs: 5’-CCCGTCGAGCTGCCAATWCTGA-3’; reverse primer cts-Rs: 5’-ATCTCGCACGGSGTRTTGAACATC-3’) (Berge et al. 2014; Sarkar et al. 2004). 409-bp cts gene sequences were deposited in the GenBank database for seven isolates (accession no. OK105110-OK105116). Sequence of seven isolates shared 100% identity with several Pseudomonas cichorii strains within the GenBank database (accession no. KY940268 and KY940271), and the phylogenetic tree of cts genes of the seven isolates clustered with the phylogroup 11 of Pseudomonas syringae (accession no. KJ877799 and KJ878111), which was classified as P.cichorii. To satisfy Koch’s postulates, a pathogenicity test was tested by using a needle to dip a suspension of the bacterium (108 CFU/ml) and pricking three holes in the tobacco leaf. The control plants leaves were needled with sterile water. Each tobacco plant was inoculated with three leaves, and the test was repeated three times. All plants were placed in transparent plastic boxes and incubated in a greenhouse at 25 ± 3°C. The water-soaked spots appeared 24h after inoculation and quickly expanded through leaf veins. Three days after inoculation, all the inoculated leaves showed symptoms similar to those observed in the field. Control plants remained healthy. Only P. cichorii was successfully re-isolated from the lesions, confirming Koch’s postulates. Pseudomonas cichorii can infect eggplant, lettuce, tomatoand other crops, and has a wide range of hosts (Timilsina et al. 2017; Ullah et al. 2015). To our knowledge, this is the first report of P. cichorii causing leaf spot on tobacco in China.

First Report of Leaf Spot Caused by Didymella americana on Cassia nomame in China

Cassia nomame (Sieb.) Kitagawa is an annual plant in the Leguminousae family. The aerial parts of C. nomame have been used as tonic and diuretic in Korea and Japan (Syed et al. 2019). A leaf spot was observed on the leaves of a 1-year-old C. nomame landrace in Changli County (39.42°N, 119.10°E), Qinhuangdao City, Hebei Province during August to October in 2018. In many fields (n≥3), the disease incidence over 80% in the middle and late stage of plant growth. Symptoms on leaves in one field began with many small, dark necrotic spot lesions. Later, the lesions spread to round-to-oval, slightly sunken in the center, and large necrotic patches with indefinite margins. Finally, lesions coalesced and resulted in defoliation. Lesions were occasionally observed on the pods. Symptoms on the pods were initially small, dark spots and then expanded to large necrotic patches with irregular edges. Symptomatic tissues (n=32) from pods and leaves were cut into 3 to 8 mm2 squares, surface disinfested with 75% ethanol for 10 s, rinsed with sterile distilled water, then placed on potato dextrose agar (PDA) at 28℃. After 3 days, ten isolates with consistent characteristics were obtained with a frequency 52.6%. The isolates on PDA were round, initially pale and had little aerial mycelium, gradually turned olive green and had dense wool-like dense aerial mycelia after 3 days. Conidia were hyaline, smooth, solitary, and elliptical. The conidia measured 5.4 to 8.2 μm × 2.5 to 3.8 μm (n=50), and has two oil bodies positioning at opposite poles. Pigmented chlamydospores were spherical or nearly pear-shaped, and solitary. Black fructifications (pycnidia) were produced profusely on PDA after subculture for 3 days. All the isolates were similar to Didymella sp. in morphology (Aveskamp et al. 2009). Choice three isolates YSGUO8 YSGGUO8-a and YSGGUO8-b to be further characterized by sequencing of the internal transcribed spacer (ITS), actin gene, and 28S large subunit of the nuclear rRNA gene (LSU) (Zhang et al. 2017). The sequences of three strains (MK836417 MZ484072 and MZ484073 for ITS, MK837604 MZ593675 and MZ593676 for actin, MK843781 MZ836208 and MZ836207 for LSU, respectively) showed 99% to 100% similarity with Didymella americana K-004 (KY070279 for ITS,KY070285 for LSU), Phoma americana CBS 256.65 (FJ426973 for ITS, FJ426871 for actin, MH870196 for LSU) and P. americana CBS 185.85 (FJ426972 for ITS, FJ426870 for actin, GU237990 for LSU) in GenBank. The fungi were identified as D. americana (formerly P. americana or Peyronellaea americana) on the basis of morphological characteristics and sequence analysis. A pathogenicity test was conducted with three times on 1-year-old C. nomame strain at the 4 to 6 compound leaf stage. Conidia were obtained from 7-day-old PDA cultures grown at 28℃ with a 12-h photoperiod. Koch’s postulates were fulfilled by spray inoculating ten healthy young plants with 106 conidia per milliliter of D. americana strain YSGUO8, and sterile water as the control. After inoculation, the plants were managed at 28℃, 60% relative humidity and a 12-h photoperiod. After 5 to 8 days, the inoculated leaves developed small and dark spots lesions similar to those observed on the leaves with initial symptoms in the field. The control leaves remained symptomless. The same fungi were re-isolated from infected leaves by morphology observation and sequence analysis, confirming Koch's postulates. D. americana has caused leaves spot on Table Beet in New York (Vaghefi et al. 2016). To our knowledge, this is the first report of D. americana causing leaf spot of C. nomame in China.

Detection and Testing Pathogenicity of Xanthomonas axonopodis pv. punicae Causing Bacterial Blight on Pomegranate

Pomegranate, one of the most important fruit crops, is constantly challenged by Bacterial blight caused by Xanthomonas axonopodispv. punicae, is a prevalent and destructive pomegranate disease. Accurate diagnosis of disease is very important to manage the disease. PCR have been widely used to detect or verify the presence of pathogen in recent decades. These molecular-based methods are rapid, accurate and sensitive for detecting pathogens. In this study, a primer set KKM5 and KKM6 was used and amplification of 491bp of gyrB gene proved the presence of Xap. The pathogenicity of the Xap was confirmed following Koch’s postulates.

Two New Bacterial Pathogens of Peanut Causing Early Seedling Decline Disease Identified in the Texas Panhandle

Peanut (Arachis hypogaea L.) is cultivated in tropical and subtropical regions of the world as an important source of oil and protein. Until now, bacterial wilt, caused by Ralstonia solanacearum, was the only known bacterial disease of peanut. In 2020, widespread incidence of poor stand establishment were observed in multiple production fields planted to the Spanish-type peanut varieties in the Texas Panhandle. The observed symptoms included seed rot, pre- and post-emergence damping-off, poor seedling vigor and death, and poorly developed root systems with little or no nodule formation. Subsequent diagnosis of symptomatic seedlings recovered two bacterial species identified by BLAST using 676 bp and 661 bp 16S rRNA fragments as a R. species and a Pantoea sp., respectively. To investigate a possible causative role of these bacteria in the observed peanut disease, the pathogenicity of the two isolates was evaluated under greenhouse conditions relying on Koch’s postulates. Cell suspensions of the two bacteria, separately and in combination, were used to inoculate seeds of a Valencia-type peanut variety with no history of the disease and found to be pathogenic on the resultant seedling plants. Symptoms that developed on the inoculated plants were similar to the symptoms initially observed in the field, including seed rot, pre- and post-emergence damping off, poor seedling vigor and root establishment. The two bacteria were also successfully recovered from inoculated and symptomatic plants, thus satisfying Koch’s postulates. Given the early onset of symptom development on affected seeds and seedlings, a seedborne origin of the disease, described here as early-decline bacterial disease of peanut, was investigated in the same batches of peanut seeds that were planted, as well as seeds later harvested in some of the affected fields. Identical bacterial species, on the basis of 16S rRNA identity, were recovered from all of the seeds evaluated indicating that the bacteria are both seedborne and seed-transmissible. Multi-locus sequence analysis (MLSA) involving six genes (dnaK, fumC, gyrB, murG, trpB, and tuf) showed that these new strains are most closely related to R. pickettii and P. dispersa, but also phylogenetically distinct. The two bacteria were designated Ralstonia sp. strain B265 and Pantoea sp. strain B270. Losses from the disease in affected fields in 2020 averaged fifty percent ($1.12 million) from a total of nine production fields. Findings from this study provide evidence for two new bacterial pathogens of peanuts capable of infecting Spanish and Valencia peanut varieties.

First report of Fusarium petroliphilum causing fruit rot of spaghetti squash in California

Spaghetti squash (Cucurbita pepo L. subsp. pepo) is a yellow-skinned squash that forms translucent spaghetti-like strands when cooked. California leads the nation in total squash production, the majority of which is grown in the San Joaquin Valley. In October of 2019, severe fruit rot of C. pepo L. subsp. Pepo (C. pepo) was observed in fruit harvested from seven cultivated fields in San Joaquin County, California. Infected fields incurred up to 30% postharvest losses. At harvest, fruit appeared healthy. After ten days in a shaded storage shed, scattered buff to tan ringed lesions extending into the flesh of infected fruits were observed. Lesions had visible sporodochia at the center that were variable in size and continued to expand in storage. Tissue (∼1 mm3) from the lesion margins of symptomatic fruit (n=8) was surface sterilized in 75 % ethanol for 1 min then 0.6% sodium hypochlorite for a minute, and aseptically transferred to half strength acidified potato dextrose agar (0.5 APDA) and incubated at 22–25 °C. Fungal colonies which grew from the pieces were light yellow, with mycelium that was flat and mucoid. Sporodochial conidia were falcate and robust with 3 to 5 septa and measured from 44.2 to 51.6 × 4.6 to 5.9 μm (average 46.3 × 5.2 μm). Aerial conidia were profuse, borne on short monophialides, ovoid to reniform, and measured 5.1 to 12.6 μm × 3.2 to 5.6 μm (average 4.2 × 6.1 μm). DNA extracted from two isolates, was amplified with primers ITS1/ITS4, and EF1-728F/EF1-986R using PCR, to obtain sequences from the internal transcribed spacer (ITS) (White 1990), and elongation factor 1α (EF1α) (Carbone et al. 1999) genetic regions. Sequences from both isolates were identical. Sequences from isolate MVAP50001827, GenBank nos. MZ081401 (ITS) and MZ102267 (EF-1α) matched 100% to sequences of representative isolates of Fusarium petroliphilum (Q.T. Chen & X.H. Fu; Short et al., 2013, MB 802539) from Cucurbita species, MF535516 (ITS) and MF580776 (EF-1α) respectively (González, V. et al. 2018). To fulfill Koch’s postulates, conidia were harvested from a culture of isolate MVAP50001827 and grown for 7 days on 0.5 APDA at room temperature (22–25 °C). A 3-cc syringe with a 25-gauge needle was used to wound and inject 200 μl of 1 × 106 conidia ml–1 into three equally spaced points 1 mm deep into the rind of C. pepo fruit (n=4). C. pepo fruit (n=4) serving as negative controls were treated similarly with 200 μl of sterile deionized water. Fruit was incubated in a growth chamber at 27 °C under 12-h diurnal cycle lighting conditions. Ten days post inoculation, lesions densely covered with white sporodochia had expanded to 7 cm diameter and 5 cm deep on average (average fruit size 31×11 cm). Twenty days post inoculation, severe fruit rot was observed. F. petroliphilum did not grow from the controls, and was successfully reisolated from the symptomatic inoculated fruits, completing Koch’s postulates. Seeds inside the inoculated fruits were completely colonized and covered in conidia. Twenty-five seeds from the source seed lot was tested for F. petroliphilum by surface sterilizing and plating onto 0.5 APDA. No F. petroliphilum grew from tested seed. Postharvest fungal diseases can affect profitability of winter squash, which is often held in storage, and sold when market prices are optimal. To our knowledge, this is the first report of Fusarium petroliphilum infecting spaghetti squash (Cucurbita pepo L. subsp. pepo) in California.