Monosporic Isolates Research Articles

First Report of Fusarium asiaticum Causing Stem Rot of Ligusticum chuanxiong in China.

Ligusticum chuanxiong (known as Chuanxiong in China) is a traditional edible-medicinal herb, which has been playing important roles in fighting against COVID-19 (Ma et al. 2020). In March 2021, we investigated stem rot of Chuanxiong in six adjacent fields (~100 ha) in Chengdu, Sichuan Province, China. The disease incidence was above 5% in each field. Symptomatic plants showed stem rot, watersoaked lesions, and blackening with white hyphae present on the stems. Twelve symptomatic Chuanxiong plants (2 plants/field) were sampled. Diseased tissues from the margins of necrotic lesions were surface sterilized in 75% ethanol for 45 s, and 2% NaClO for 5 min. Samples were then rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) at 25ºC for 72 h. Fourteen fungal cultures were isolated from 18 diseased tissues, of which eight monosporic isolates showed uniform characteristics. The eight fungal isolates showed fluffy white aerial mycelia and produced yellow pigments with age. Mung bean broth was used to induce sporulation. Macroconidia were sickle-shaped, slender, 3- to 5-septate, and averaged 50 to 70 μm in length. Based on morphological features of colonies and conidia, the isolates were tentatively identified as Fusarium spp. (Leslie and Summerell 2006). To identify the species, the partial translation elongation factor 1 alpha (TEF1-α) gene was amplified and sequenced (O'Donnell et al. 1998). TEF1-α sequences of LCSR01, LCSR02 and LCSR05 isolates (GenBank nos. MZ169386, MZ169388 and MZ169387) were 100%, 99.72% and 99.86% identical to that of F. asiaticum strain NRRL 26156, respectively. The phylogenetic tree based on TEF1-α sequences showed these isolates clustered with F. asiaticum using Neighbor-Joining algorithm. Furthermore, these isolates were identified using the specific primer pair Fg16 F/R (Nicholson et al. 1998). The results showed these isolates (GenBank nos. MZ164938, MZ164939 and MZ164940) were 100% identical to F. asiaticum NRRL 26156. Pathogenicity test of the isolate LCSR01 was conducted on Chuanxiong. After wounding Chuanxiong stalks and rhizomes with a sterile needle, the wounds were inoculated with mycelia PDA plugs. A total of 30 Chuanxiong rhizomes and stalks were inoculated with mycelia PDA plugs, and five mock-inoculated Chuanxiong rhizomes and stalks served as controls. After inoculation, the stalks and rhizomes were kept in a moist chamber at 25°C in the dark. At 8 days post inoculation (dpi), all inoculated stalks and rhizomes exhibited water-soaked and blackened lesions. At 10 dpi, the stalks turned soft and decayed, and abundant hyphae grew on the exterior of infected plants, similar to those observed in the field. No disease symptoms were observed on the control plants. The pathogen was re-isolated from the inoculated tissues and the identity was confirmed as described above. Ten fungal cultures were re-isolated from the 10 inoculated tissues, of which nine fungal cultures were F. asiaticum, fulfilling Koch's postulates. To our knowledge, this is the first report of F. asiaticum causing stem rot of Chuanxiong in China. Chuanxiong has been cultivated in rotation with rice over multiple years. This rotation may have played a role in the increase in inoculum density in soil and stem rot epidemics in Chuanxiong. Diseased Chuanxiong may be contaminated with the mycotoxins produced by F. asciaticum, 3-acetyldeoxynivalenol or nivalenol, which may deleteriously affect human health. Therefore, crop rotations should be considered carefully to reduce disease impacts.

Evaluation of sensitivity of apple scab pathogen to difenoconazole using the discriminatory dose technique

The most serious disease of the apple tree in all areas of its growth is scab. In the integrated apple tree protection system, the main method is chemical. However, the use of chemical fungicides is characterized by the risk of developing resistance to them by pathogen. The sensitivity of 118 monospore isolates of Venturia inaequalis was studied from three orchards of Jeromine, Reinette Simirenko and Gala cultivars, differing in the frequency of application of difenoconazole. Sensitivity was determined using the discriminatory dose technique (0.01 mg/l of active substance) in terms of RG, the relative growth of the mycelium. RG was expressed as the degree of change in mycelium growth in a nutrient medium with fungicide relative to the control variant in percent. All pathogen populations studied differed significantly in mean RG values. Populations treated three times per season with difenoconazole had higher RG values compared to populations treated two times. From Gala orchard, for some isolates, a stimulating effect of a discriminatory dose of difenoconazole on their growth was observed, that is, a hormesis effect was manifested. The proportion of isolates with RG values above the cutoff value, which was 84 and 100 % for the Reinette Simirenko and Gala orchards, may indicate that the pathogen populations studied are resistant, and in these orchards, there may be a decrease in the effectiveness of protection against apple scab. The discriminatory dose technique allowed us to objectively and promptly assess the sensitivity of V. inaequalis populations from orchards with varying intensity use of difenoconazole.

First Report of Passion Fruit Anthracnose Caused by Colletotrichum constrictum.

Passion fruit (Passiflora edulis) is widely cultivated in tropic and subtropic regions. Because of its unique and intense flavour and high acidity, passion fruit juice concentrate is used in making delectable sauces, desserts, candy, ice cream, sherbet, or blending with other fruit juices. Anthracnose of passion fruit is favored by frequent rainfall and average temperatures above 27°C. In August 2018, anthracnose on passion fruit was observed in commercial plantings in Lincang, Yunnan, China (23.88 N, 100.08 E). Symptoms included lesions of oval to irregular shapes with brown to dark brown borders. Infection covered most of the fruit surface with pink-to-dark sporulation as reported by Tarnowski and Ploetz (2010). A conidial mass from an individual sorus observed on an infected fruit was isolated and cultured on potato dextrose agar (PDA) supplemented with 50 μg ml-1 of streptomycin. From a single microscopic field, two monospore isolates were dissected using a sterile needle, subcultured, and referred to as BXG-1 and BXG-2. Morphological characters including conidia colour, size, and shape were similar between the two isolates. Conidia were aseptate and cylindrical with apex and rounded base. Conidial length ranged from 12.3 to 16.1 µm (avg. 13.5) and width ranged from 5.5 to 6.2 µm (avg. 5.7). Morphologic data were consistent with Colletotrichum constrictum (Damm et al., 2012). To further confirm the fungal species, the ribosomal internal transcribed spacer (ITS), partial sequences of actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin 2 (TUB2) were amplified and sequenced. Primers and PCR amplification were described by Damm et al. (2012). The sequences were compared to type sequences in GenBank. The results showed the ITS (GenBank accession MW828148 and MW828149), ACT (MW855882 and MW855883), CHS-1 (MW855884 and MW855885), GAPDH (MW855886 and MW855887), and TUB2 (MW855888 and MW855889) sequences of the isolates BXG-1 and BXG-2 were 98% identical with sequence data from strain CBS:128504 of C. constrictum. A maximum likelihood tree was constructed using MEGA-X version 10.1.6 (Kumar et al., 2018) based on a combined dataset of the ITS, ACT, CHS-1, GAPDH, and TUB2 sequences of BXG-1 and BXG-2, and those of 18 Colletotrichum spp. previously deposited in GenBank (Damm et al., 2012). The phylogenetic analysis showed that BXG-1 and BXG-2 belong to the C. constrictum clade. Based on morphology and DNA sequencing, BXG-1 and BXG-2 were identified as C. constrictum. To verify pathogenicity, passion fruit were sprayed with a suspension of 1 × 105 conidia ml-1. Control fruit were sprayed with sterilized water. After inoculation, fruit were incubated in an Artificial Climate Box at 27°C and 80% RH. Necrotic symptoms appeared 8 days after inoculation and were similar to those observed on fruit form the field. The pathogen was reisolated from lesions thus fulfilling Koch's postulates. C. constrictum has been reported to cause anthracnose of citrus from Australia (Wang et al., 2021) and mango from Italy (Ismail et al., 2015). To our knowledge, this is the first report of C. constrictum causing anthracnose on passion fruit worldwide, and these data will provide useful information for developing effective control strategies.

First Report of Brown Spot Needle Blight on Pinus thunbergii Parl. Caused by Aureobasidium pullulans in China.

Pinus thunbergii Parl., known as black pine, is widely distributed all over China. This pine variety can prevent soil desertification and promote soil conservation and is excellent for constructing fast-growing forests and shelter belts. The timber of this species can be used for infrastructure construction and furniture production. In August 2020, needle blight symptoms were found on several trees of black pine in Sichuan Province, China. Further surveys showed that these symptoms are common while the disease incidence is less than 30% which indicated the severity of the disease is mild. The tips of old needles first turn grayish green and developed into brown bands ranging from 1 to 2 mm. To determine the pathogen, 20 needle samples with typical symptoms were disinfected with 75% alcohol, and sections of the tissue were cut from joints of diseased and healthy tissues (visually healthy) with a sterilized scalpel, surface sterilized for 45 seconds in 75% alcohol, soaked for 90 seconds in 1.5% NaCIO, rinsed in sterilized water and dried. Small cut tissues were placed on potato dextrose agar (PDA) at 25℃ for 10 days. Pure cultures were obtained by monosporic isolation. The colonies initially appeared white to cream, yeast-like, and later turned to pink and remained at least 10 days. Conidia were hyaline, smooth-walled, single-celled, and ellipsoidal with variable shape and size, 7.5 to 16 × 3.5 to 7 µm (Zalar et al. 2008). DNA was extracted from the mycelium of the isolate by the cetyltriethylammonium bromide (CTAB) method and amplified through polymerase chain reaction (PCR) with the internal transcribed spacer (ITS) region of rDNA and partial β-tubulin genes of a representative isolate (SC05) were amplified using the ITS1/ITS4 and Bt2a/Bt2b primer pairs, respectively(Wu et al. 2017). The sequences submitted to GenBank (Accession Nos. MW228368 for ITS and MW256762 for β-tubulin) showed high similarity with BLAST sequences of Aureobasidium pullulans (ITS, KR704881 [100%]; β-tubulin, MT671934 [99.49%]). For the pathogenicity test, a conidial suspension was prepared with a concentration of 2.0 × 107 conidia/ml. The suspension was sprayed onto 3 annual seedlings' needles, and the control was sprayed with sterile water. Inoculated and non-inoculated plants were kept in humid chambers in a glasshouse. After 10 days, typical symptoms appeared on inoculated needles, whereas control needles remained symptomless. The fungus, A. pullulans, was reisolated from those lesions, confirming Koch's postulates. No symptoms were observed on control plants. Aureobasidium pullulans, a ubiquitous saprophytic fungus on many fruits and very rarely reported to cause disease on pine needles. Only reported invasion of Ozone-injured needles in P. strobus (Costonis and Sinclair 1972) and needles damaged by acid rain in P. sylvestris (Ranta 1990). To our knowledge, this is the first report of brown spot needle blight on P. thunbergii caused by A. pullulans in China. The disease represents a threat to pine manufactures and more research on the pathogenesis and management is needed. .

First report of Anthracnose on Peach Fruit Caused by Colletotrichum siamense in Uruguay.

Peach (Prunus persica L.) is an economically important deciduous fruit crop in Uruguay. Anthracnose caused by species of the genus Colletotrichum is one of the major diseases in peach production, originating significant yield losses in United States (Hu et al. 2015), China (Du et al. 2017), Korea (Lee et al. 2018) and Brazil (Moreira et al. 2020). In February 2017, mature peach fruits cv. Pavia Canario with symptoms resembling anthracnose disease were collected from a commercial orchard located in Rincon del Colorado, Canelones, in the Southern region of Uruguay. Symptoms on peach fruit surface were characterized as circular, sunken, brown to dark-brown lesions ranging from 1 to 5 cm in diameter. Lesions were firm to touch with wrinkled concentric rings. All lesions progressed to the fruit core in a V-shaped pattern. The centers of the lesions were covered by orange conidial masses. Monosporic isolates obtained from the advancing margin of anthracnose lesions were grown on PDA at 25ºC and 12h photoperiod under fluorescent light. The representative isolates DzC1, DzC2 and DzC6 were morphologically and molecularly characterized. Upper surface of colonies varied from white or pale-gray to gray and on the reverse dark-gray with white to pale-gray margins. Conidia were cylindrical, with both ends predominantly rounded or one slightly acute, hyaline and aseptate. The length and width of conidia ranged from 9.5 to 18.9 µm (x ̅=14.1) and from 3.8 to 5.8 µm (x ̅=4.6), respectively. The ACT, βTUB2, GAPDH, APN2, APN2/MAT-IGS, and GAP2-IGS gene regions were amplified and sequenced with primers ACT-512F/ACT-783R (Carbone and Kohn, 1999), BT2Fd/BT4R (Woudenberg et al. 2009), GDF1/GDR1 (Guerber et al. 2003), CgDLR1/ColDLF3, CgDLF6/CgMAT1F2 (Rojas et al. 2010) and GAP1041/GAP-IGS2044 (Vieira et al. 2017) respectively and deposited in the GenBank database (MZ097888 to MZ097905). Multilocus phylogenetic analysis revealed that Uruguayan isolates clustered in a separate and well supported clade with sequences of the ex-type (isolate ICMP 18578) and other C. siamense strains (isolates Coll6, 1092, LF139 and CMM 4248). To confirm pathogenicity, mature and apparently healthy peach fruit cv. Pavia Canario were inoculated with the three representative isolates of C. siamense (six fruit per isolate). Fruit were surface disinfested with 70% ethanol and wounded with a sterile needle at two equidistant points (1 mm diameter x 1 mm deep). Then, fruit were inoculated with 5 µl of a spore suspension (1×106 conidia mL-1) in four inoculation points per fruit (two wounded and two unwounded). Six fruit mock-inoculated with 5 µl sterile water were used as controls. Inoculated fruit were placed in moist chamber and incubated at 25°C during 10 days. Anthracnose lesions appeared at 2 and 4 days after inoculation in wounded and unwounded points, respectively. After 7 days, disease incidence was 100% and 67% for wounded and unwounded fruit, respectively. The control treatment remained symptomless. The pathogens were re-isolated from all lesions and re-identified as C. siamense. C. siamense was previously reported in South Carolina causing anthracnose on peach (Hu et al. 2015). To our knowledge, this is the first report of anthracnose disease on peach caused by C. siamense in Uruguay. Effective management strategies should be implemented to control anthracnose and prevent the spread of this disease to other commercial peach orchards.

Cercospora species cause pink spot disease on guava fruit in Brazil

AbstractGuava producers in Brazil have reported the occurrence of small, pink spot lesions on the surface of fruits in orchards. Symptomatic fruits were harvested from commercial guava orchards, and 18 monosporic isolates were obtained. All tested isolates were pathogenic to guava fruit and showed colony and conidial characteristics indicative of cercosporoid fungi. Phylogenetic analysis of the concatenated gene sequences showed that all isolates belonged to the genus Cercospora and clustered into four haplotypes. Conidia released 3–5 germ tubes, and penetration occurred through the stomata. All isolates produced cercosporin. Histopathological analyses showed that hyphal growth occurred intercellularly and that there was a collapse of epidermal and subepidermal cells in the injured areas. In addition, the oil cavities in the lesioned tissue had large numbers of lipid droplets compared with the nonlesioned tissue. This is the first report of Cercospora spp. as the causal agent of pink spot disease of guava fruit in Brazil.

First report of leaf spot disease caused by Corynespora cassiicola on American sweetgum (Liquidambar styraciflua L.) in China.

American sweetgum (Liquidambar styraciflua L.) is a forest plant native to North America, which has been introduced into other countries due to its ornamental and medicinal values. In June 2019, symptoms of leaf spots on sweetgum were observed in a field (5 ha) located in Xuzhou, Jiangsu Province, China. On this field, approximately 45% of 1,000 trees showed the same symptoms. Symptoms were observed showing irregular or circular dark brown necrotic lesions approximately 5 to 15 mm in diameter with a yellowish margin on the leaves. To isolate the pathogen, diseased leaf sections (4×4mm) were excised from the margin of the lesion, surface-sterilized with 0.1% NaOCl for 90 s, rinsed 4 times in sterile distilled water, air dried and then transferred on potato dextrose agar (PDA) medium at 25°C in the dark. Pure cultures were obtained by monospore isolation after subculture. Ten purified isolates, named FXI to FXR, were transferred to fresh PDA and incubated as above to allow for morphological and molecular identification. After 7 days, the aerial mycelium was abundant, fluffy and exhibited white to greyish-green coloration. The conidia were dark brown or olive, solitary or produced in chains, obclavate, with 1 to 15 pseudosepta, and measured 45 to 200µm  10 to 18µm. Based on morphological features, these 10 isolates were identified as Corynespora cassiicola (Ellis et al. 1971). Genomic DNA of each isolate was extracted from mycelia using the cetyltrimethylammonium bromide (CTAB) method. The EF-1α gene and ITS region were amplified and sequenced with the primer pairs rDNA ITS primers (ITS4/ITS5) (White et al. 1990) and EF1-728F/EF-986R (Carbone et al.1999) respectively. The sequences were deposited in GenBank. BLAST analysis revealed that the ITS sequence had 99.66% similarity to C. cassiicola MH255527 and that the EF-1α sequence had 100% similarity to C. cassiicola KX429668A. maximum likelihood phylogenetic analysis based on EF-1α and ITS sequences using MEGA 7 revealed that ten isolates were placed in the same clade as C. cassiicola (Isolate: XQ3-1; accession numbers: MH572687 and MH569606, respectively) at 98% bootstrap support. Based on the morphological characteristics and phylogenetic analyses, all isolates were identified as C. cassiicola. For the pathogenicity test, a 10 µl conidial suspension (1×105 spores/ml) of each isolate was dripped onto healthy leaves of 2-year-old sweetgum potted seedlings respectively. Leaves inoculated with sterile water served as controls. Three plants (3 leaves per plant) were conducted for each treatment. The experiment was repeat twice. All seedlings were enclosed in plastic transparent incubators to maintain high relative humidity (90% to 100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. After 10 days, leaves inoculated with conidial suspension of each isolate showed symptoms of leaf spots, similar to those observed in the field. Control plants were remained healthy. In order to reisolate the pathogen, surface-sterilized and monosporic isolation was conducted as described above. The same fungus was reisolated from the lesions of symptomatic leaves, and its identity was confirmed by molecular and morphological approaches, thus fulfilling Koch's postulates. Chlorothalonil and Boscalid can be used to effectively control Corynespora leaf spot (Chairin T et al.2017). To our knowledge, this is the first report of leaf spot caused by C. cassiicola on L. styraciflua in China.

First Report of a Leaf Blight Caused by Pyricularia pennisetigena on Cenchrus echinatus in Paraguay.

The genus Pyricularia contains several fungal species known to cause diseases on plants in the Poaceae family (Klaubauf et al. 2014; Wang et al. 2019). While sampling for P. oryzae during March-2015 and April-2018, common weed Cenchrus echinatus L. was observed with leaf lesions in and around experimental wheat fields in the departments of Canindeyú and Itapúa. C. echinatus samples from both locations displayed similar leaf lesions, varying from small light brown pinpoint to elliptical brown lesions with greyish center. Symptomatic leaves were surface disinfested and cultured on potato dextrose agar (PDA) amended with 1% gentamicin at 25°C. Two monosporic isolates were obtained, one from Itapúa (ITCeh117) and the other from Canindeyú (YCeh55). The isolates were subsequently grown on oatmeal agar (OA) and PDA under a 12-h photoperiod at 25°C and evaluated after ten days for colony diameter, sporulation, macroscopic and microscopic features. Colonies on OA reached up to 4.8 cm diameter and were light grey, whereas colonies on PDA reached up to 5.3 cm diameter and were brown with grey centers, with cottony mycelium and broad white rims. Mycelium consisted of smooth, hyaline, branched, septate hyphae 4-4.5 µm diameter. Conidiophores were erect, straight or curved, unbranched, medium brown and smooth. Conidia were solitary, pyriform, pale brown, smooth, granular, 2-septate, 32-33 × 9-10 μm; truncated with protruding hilum and varied in length from 1.0 to 1.5 μm and diameters from 2.0 to 2.2 μm. Both isolates were similar and identified as Pyricularia pennisetigena, according to morphological and morphometric characteristics (Klaubauf et al. 2014). Subsequently, genomic DNA was extracted from each isolate using the primers described in Klaubauf et al. (2014) to amplify and sequence the internal transcribed spacers (ITS), partial large subunit (LSU), partial RNA polymerase II large subunit gene (RPB1), partial actin gene (ACT), and partial calmodulin gene (CAL). Sequences from each isolate (YCeh55/ITCeh117) were deposited in GenBank with the following submission ID for ITS: MN947521/MN947526, RPB1: MN984710/MN984715, LSU: MN944829/MN944834, ACT: MN917177/MN917182, and CAL: MN984688/MN984693. Phylogenetic analysis was conducted using the software Beast v1.10.4. The results obtained from the concatenated matrix of the five loci placed these isolates in the P. pennisetigena clade. To confirm pathogenicity, each isolate was adjusted to 5×104 conidia/ml of sterile water and C. echinatus plants were sprayed with the conidial suspension for isolate YCeh55, ITCeh117 or sterile water using an oilless airbrush sprayer until runoff. The three treatments were kept in the greenhouse at 25-28°C and about 75% relative humidity under natural daylight. Each treatment included three to five inoculated plants and 10 leaves were evaluated per treatment. Symptoms were observed 8-15 days after inoculation and were similar to those originally observed in the field for both isolates, whereas the control plants remained asymptomatic. P. pennisetigena was re-isolated from the inoculated leaves fulfilling Koch's postulates. To our knowledge, this is the first report of leaf blight on C. echinatus caused by P. pennisetigena in Paraguay. The occurrence of P. pennisetigena in the region and its ability to infect economically important crops such as wheat and barley (Klaubauf et al. 2014; Reges et al., 2016, 2018) pose a potential threat to agriculture in Paraguay.

First Report of Wilt of European hornbeam (Carpinus betulus L.) Caused by Fusarium oxysporum in China.

European hornbeam (Carpinus betulus L.) has been used as an important ornamental species for urban landscaping since the Italian Renaissance (Rocchi et al. 2010). In May 2019, 15% of 3000 C. betulus trees with wilted leaves and root rot were observed in a field (about 26 ha) in Pizhou, Jiangsu Province, China. Internal discoloration of the stem began with brown to black discoloration of the vascular system and gradually spread to inward areas. Roots and stems from symptomatic plants were washed free of soil, surface sterilized with 0.8% NaOCl, rinsed three times in sterile H2O, and blotted dry with a paper towel. Small segments (0.5-cm-long) were cut from the discolored vascular tissues, and then put on potato dextrose agar (PDA) at 25°C in darkness. After 4 days, fungal colonies were observed on the PDA. Pure cultures were obtained by monosporic isolation, and 9 morphologically similar fungal isolates (EJ-1 to EJ-9) were obtained. All purified cultures were incubated on PDA at 25°C in darkness as the initial isolation. Colonies of the 9 isolates on PDA displayed entire margins and showed abundant pink aerial mycelia initially and turned to light violet with age. Microconidia were elliptical or oval in shape, 0 septate, (5.2-)8.7(-12.5) × (3.5-)3.6(-5.5) µm. Macroconidia were falciform, 0-4 septate, and straight to slightly curved with a notched foot cell, (17.1-)20.5(-28.4) × (3.8-)4.1(-4.6) µm. These morphological characteristics resemble Fusarium oxysporum (Leslie and Summerell 2006). Genomic DNA of each isolate was extracted from mycelia using a CTAB method (Mo¨ller et al. 1992). The RPB2, TEF1 and cmdA genes were amplified and sequenced with the primers 5f2/7c (Liu et al. 2000), EF-1Ha/EF-2Tb (Carbone and Kohn 1999) and Cal228F/CAL2Rd (Groenewald et al. 2013), respectively. The sequences were deposited in GenBank (Table 1). A maximum likelihood phylogenetic analysis based on RPB2, TEF1 and cmdA sequences using MEGA7 revealed that the isolates were placed in the F. oxysporum species complex with 98% bootstrap support. Based on the morphological and molecular characters, all 9 isolates were identified as F. oxysporum. A pathogenicity experiment was conducted using 30 2-year-old C. betulus seedlings potted in sterile peat, 27 for inoculation (3 replicate plants per isolate) and 3 for a negative control. The treated plants were planted in the peat mixed with 50 ml of a conidial suspension of each isolate respectively. The negative control was inoculated with sterilized water. Conidia were harvested from colonized plates of PDA using sterilized water and adjusted to a concentration of 1×107 conidia/ml. All 30 seedlings were incubated in a greenhouse at 25°C with a relative humidity of 80% and a 12-h photoperiod. The inoculated seedlings displayed wilt symptoms within 30 to 40 days, and eventually died within 75 to 85 days after inoculation. Control plants remained symptomless. F. oxysporum was successfully reisolated from the vascular tissues of symptomatic plants, and sequences of RPB2, TEF1 and cmdA of re-isolates matched those of the original isolates. No pathogen was isolated from the tissues of control plants. The experiment was repeat twice with the similar results, fulfilling Koch's postulates. F. oxysporum is an important soil-borne pathogen and can cause disease in many economic plants, such as yellowwood (Graney et al. 2016), hickory (Zhang et al. 2015) and larch (Rolim et al. 2020). To our knowledge, this is the first report of wilt on C. betulus caused by F. oxysporum in China.

First Report of Leaf Spot on Industrial Hemp (Cannabis sativa L.) Caused by Alternaria alternata in China.

Industrial hemp is an economically important plant with traditional uses for textiles, paper, building materials, food and medicine (Li 1974; Russo et al. 2008; Zlas et al. 1993). In August 2020, an estimated 80% of the industrial hemp plants with leaf spots were observed in greenhouse in Minzhu town, Harbin City, Heilongjiang Province, China (45.8554°N, 126.8167°E), resulting in yield losses of 20%. Leaf symptoms began as small spots on the upper surface of leaves and gradually developed into brown spots with light yellow halos. These irregular spots expanded gradually and eventually covered the entire leaf; the center of the spots was easily perforated. To identify the pathogen, 20 diseased leaves were collected, and small sections of (3 to 5 mm) were taken from the margins of lesions of infected leaves. The pieces were sterilized with 75% alcohol for 30 s, a 0.1% mercuric chloride solution for 1 min, and then rinsed three times with sterile water. Samples were then cultured on potato dextrose agar at 28℃ in darkness for 4 days. A single-spore culture was obtained by monosporic isolation. Conidiophores were simple or branched, straight or flexuous, brown, and measured 22 to 61 μm long × 4 to 5 μm wide (n = 50). Conidia were solitary or in chains, brown or dark brown, obclavate, obpyriform or ellipsoid. Conidia ranged from 23 to 55 μm long × 10 to 15 μm wide (n = 50) with one to eight transverse and several longitudinal septa. For molecular identification (Jayawardena et al. 2019), genomic DNA of pathogenic isolate (MZ1287) was extracted by a cetyltrimethylammonium bromide protocol. Four gene regions including the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosplate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1) and RNA polymerase II beta subunit (RPB2) were amplified with primers ITS1/ITS4, GDF1/GDR1, EF1-728F/EF1-986R and RPB2-5F/RPB2-7cR, respectively (White et al. 1990). Resulting sequences were deposited in GenBank with accession numbers of MW272539.1, MW303956.1, MW415414.1 and MW415413.1, respectively. A BLASTn analysis showed 100% homology with A. alternata (GenBank accession nos. MN615420.1, MH926018.1, MN615423.1 and KP124770.1), respectively. A neighbor-joining phylogenetic tree was constructed by combining all sequenced loci in MEGA7. The isolate MZ1287 clustered in the A. alternata clade with 100% bootstrap support. Thus, based on morphological (Simmons 2007) and molecular characteristics, the pathogen was identified as A. alternata. To test pathogenicity, leaves of ten healthy, 2-month-old potted industrial hemp plants were sprayed using a conidial suspension (1×106 spores/ml). Control plants were sprayed with sterile water. All plants were incubated in a greenhouse at 25℃ for a 16 h light and 8 h dark period at 90% relative humidity. The experiment was repeated three times. After two weeks, leaf spots of industrial hemp developed on the inoculated leaves while the control plants remained asymptomatic. The A. alternata pathogen was re-isolated from the diseased leaves on inoculated plants, fulfilling Koch's postulates. Based on morphology, sequencing, and pathogenicity test, the pathogen was identified as A. alternata. To our knowledge, this is the first report of A. alternata causing leaf spot disease of industrial hemp (Cannabis sativa L.) in China and is worthy of our attention for the harm it may cause to industrial hemp production.

First Report of leaf spot disease caused by Colletotrichum gloeosporioides on Chaenomeles sinensis in China.

Chaenomeles sinensis is a shrub or small arbor of the genus Chaenomeles in Rosaceae, which is widely planted in China. It is a kind of garden ornamental plant and has high economic value. Since 2020, a leaf disease occurred on the foliage of C. sinensis at the campus of Nanjing Forestry University, Nanjing, China. After investigating, C. sinensis was found with leaf spot disease at a 100% infection rate, which causing gigantic ornamental loss. Leaf spots are round to irregular distributing on the leaves, in addition, the color of spots is brown. There are yellow halos on the edge of the lesion. Small leaf tissues (3 to 4 mm2) from lesion margins were surface sterilized with 75% ethanol for 30s and then rinsed with sterile dH2O for three times. Afterwards, placed on potato dextrose agar (PDA) at 25°C. Pure cultures were obtained by monosporic isolation, and a representative isolate (NJTJ.1) was obtained. When cultured on PDA, the colony of NJTJ.1 was white and cottony. On the reverse side, the color of colony nearly light yellow. The colony were placed in the liquid Carboxymethyl cellulose (CMC) medium. After culturing for 24h in a shaker at 25℃ and 150rmp/min, the spore liquid was taken by us. The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and measured 15.1 to 23.6× 5.4 to 7.9 µm (n =30). Appressoria were one-celled, brown, thick-walled, ellipsoidal, and measured 7.7 to 13.8 × 6.4 to 10.3 µm (n =30). The morphological characteristics of NJTJ.1 fitted with the description of the Colletotrichhum gloeosporioides complex (Weir et al., 2012). For accurate identification, the internal transcribed spacer (ITS), and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS-1) were amplified with primers ITS1/ITS4, GDF/GDR, ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al, 2019), respectively. The sequences were deposited in GenBank [Accession Nos.MT984264, MW030495 and MW030496 to MW030497 for NJTJ.1]. A Blast search of GenBank showed that ITS, GAPDH, ACT and CHS-1 sequences of NJTJ.1 were 99%, 99%, 100% and 100% identical to those of C. gloeosporioides (MH571757.1 ,KY995355.1 , MN058143.1 and MN313581.1). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate NJTJ.1 clustered in the C. gloeosporioides clade with 99% bootstrap support. The pathogenicity of the NJTJ.1 was verified both on detached and living leaves. The detached leaves were inoculated with 5-mm mycelial plugs cut from the edge of 6-day old cultures on PDA and 20 μL of spore suspension (106 conidia/mL) and each treatment had 5 replicates. Controls were treated with sterile dH2O. The inocula were placed at a distance of 2 to 3 cm on the leaves which were wounded with a sterile needle. All of them were placed in 20-cm dishes on wet filter paper at 25°C. After 5 days, all the inoculated points showed lesions which were similar to those outdoor observed. Whereas, controls were asymptomatic.At the same time, the plugs of C. gloeosporioides were inoculated on living leaves.After 7 days, the leaves which were inoculated also appeared lesions. This is the first report of C. gloeosporioides causing leaf blotch on Chaenomeles sinensis in China. These data will help develop effective strategies for managing this disease.

Dried fruits marketed in Russian: toxigenic mold contamination

Introduction. Dried fruits are a valuable source of dietary fibre, many vitamins and minerals in the population’s diet. However, the high content of readily available carbohydrates makes this type of product vulnerable to mould contamination. The greatest danger among which are toxigenic species. But there is practically no scientific information about the contamination presented on the Russian market dried fruits with moulds producing mycotoxins. That does not allow judging about this aspect of food safety. Materials and methods. Contamination with moulds and bacteria of 57 samples of dried fruits of 7 species popular in Russia was studied using cultural methods of analysis. Monospore isolates of moulds were isolated from dried fruits; in vitro mycotoxins production studied; by UHPLC-MS / MS analyzed mycotoxins in the multidetection mode. Results and discussion. In general, the microbial contamination of dried fruits was low: 87.7% of the samples met the established microbiological standards, in most cases, moulds caused it. At the same time, the highest frequency and levels of contamination were found in dates. Aspergillus sp. dominated in the micoflora of all types of dried fruits. Among the isolated 33 strains of moulds, 45.5% turned out to be toxigenic and, in vitro, were capable of biosynthesis of significant amounts of several types of mycotoxins, including emergent mycotoxins. Fumonisin- and ochratoxin-producing activities have been found in Aspergillus strains of the Nigri section. In model experiments, the accumulation of mycotoxins in individual strains exceeded the level normalized in grain products, including (in μg/kg): for aflatoxins B1 - more than 32000 and B2 - 3230; fumonisin B2 - more than 3100; ochratoxin A up to 4.3; for emergent accumulation reached: sterigmatocystin up to 6218220 and citreoviridine - 153. Conclusion. Moulds are the main type of microflora that contaminates dried fruits. The ability of mould isolates from dried fruits to form mycotoxins has been established, among which highly toxigenic strains have been identified. This indicates the presence of a potential risk of contamination of this type of food with unregulated mycotoxins and a possible increase in their content in the diets of consumers. The results obtained substantiate the need for extensive monitoring of mycotoxin producers in dried fruits. This is important for predicting the risk of toxin formation and identifying the relationship of specific mycotoxins with certain types of dried fruits. The presence of toxigenic activity of moulds isolated from dried fruits has been shown in Russia for the first time.

Detection and characterization of QoI resistance in Pyrenophora tritici‐repentis populations causing tan spot of wheat in Argentina

AbstractTan spot, caused by the fungus Pyrenophora tritici‐repentis (Ptr), is a disease that has become more prevalent and intense in wheat crops in Argentina in recent years. Failure to control the disease with strobilurin fungicides, which were once effective, has been observed in different zones where wheat is grown. However, whether or not true resistance is present in the pathogen population in the region is not scientifically confirmed. This study evaluated the sensitivity of numerous Ptr isolates to representative QoI fungicides used in Argentina through in vitro and in planta assays, as well as through molecular analysis. Eighty‐two monosporic isolates obtained in different locations in the north and south of Buenos Aires province in 2014, 2016, and 2018 were tested to determine sensitivity to selected QoI fungicides in conidial germination and mycelial inhibition assays, as well as in molecular analysis. Conidial germination was not inhibited at 1 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin. On the other hand, mycelial growth was inhibited by 59%, 56%, and 86% at 100 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin, respectively. The molecular analysis detected the G143A mutation in the cytb gene of all the 82 Ptr isolates, but the F129L and G137R substitutions were not present. This study documents the G143A mutation conferring QoI resistance in Ptr in South America. The findings of this study are key for future decisions regarding use of fungicide and rotation in the region.

First Report of Brown Rot of fruit on Vitis amurensis Caused by Monilinia polystroma in China.

Vitis amurensis Rupr. (Amur grape) is a wild grape genetic resource widely distributed in Heilongjiang, Jilin, Liaoning, and Inner Mongolia, among other places in China (Song et al. 2009) and the Russian Far East and Korean Peninsula. In September 2018, brown rot symptoms were observed at ripening stage on the fruits of a 5-year-old Amur grapevine germplasm resources nursery of the cultivar 'Beibinghong' and a few Russian resources in Zuojia Town, Jilin City, Jilin Province, China. The diseased fruit surface became brown with soft rot and produced buff to brownish-grey sporodochia with conidia. Around 180 plants of 'Beibinghong' were examined which had 8 % incidence. Forty five samples were collected from symptomatic fruits of 15 randomly sampled 'Beibinghong' grape clusters, cut into 5-mm2 pieces of diseased tissue, surface sterilized with 1% NaOCl for 2 min, rinsed three times with sterile water, dried on sterilized filter paper, and plated on potato dextrose agar (PDA). Thirteen monosporic isolates were obtained using the single-spore isolation method with incubation at 25°C and a 12-h light/12-h dark cycle. The average colony diameter was 46-49 mm after 4 days of culture on PDA. Colonies were white to grayish with even margins. Irregular black stromatal crusts were observed on the reverse side of dishes 10 days after inoculation. Conidial spores were produced when cultured on cherry agar at 25°C under near-ultravolet light. Spores were single-celled and hyaline, limoniform or ellipsoid, and were produced in branched monilioid chains, 12-22 × 8-13 µm (mean: 15.4 ± 1.03 × 9.01 ± 0.72 µm, n = 50). When conidia were cultured on water agar at 25°C for 18 h, the germ tubes were straight, 700-1,000 µm long, and often with two germ tubes per conidium. Morphological characteristics were consistent with those of Monilinia polystroma (van Leeuwen et al. 2002). To confirm the species identification, two DNA regions of the selected isolate 'VAMPWYZSH8' were amplified by polymerase chain reaction (PCR) and sequenced: the internal transcribed spacer region (ITS) was generated using primers ITS1/ ITS4 (Munda 2015) and β-tubulin (TUB2) was amplified using primers Bt2a/Bt2b (Zhu et al. 2016). A BLAST analysis of the nucleotide sequence of the PCR products revealed 100% identity with two M. polystroma sequences in the NCBI GenBank (KJ814976 for ITS, KR778970 for TUB2). Our sequences were deposited in GenBank with accession nos. MT038413 for ITS and MT038414 for TUB2. On the basis of these results, the isolate was identified as M. polystroma. To confirm pathogenicity, 78 fresh and healthy bunches of 'Beibinghong' grapes at ripening were collected, surface disinfected by immersion in 1% NaOCl for 1 min, rinsed three times with sterile water, then allowed to air dry. Under dry aseptic conditions, the fruits were inoculated using the pin prick method. Each wound was inoculated with 10 μl conidial suspension (106 spore ml-1) and incubated at 25°C with about 90% relative humidity and natural light. Inoculation with water was used as control and the experiment was repeated three times. After a 10-day incubation, typical symptoms of brown rot developed on inoculated fruits, while control fruits were symptomless. The fungus was consistently re-isolated only from diseased fruits and showed the same morphological characteristics as the original isolates, thus fulfilling Koch's postulates. This is the first report of M. polystroma on V. amurensis in China. The resulting disease decreases fruit quality and yield, necessitating the development of effective control measures.

English

Angular leaf spot (ALS) is one of the most severe diseases of common bean. However, there is as yet no standardization of the phenological stage, inoculum concentration, diagrammatic scale (DS), and day of assessment for screening ALS resistance in bean lines. The present study aimed to evaluate the potential for assessment of ALS severity in common beans using the Leaf Doctor app and inoculation at the V2 phenological stage under two inoculation concentrations. The experiment was carried out in a completely randomized design in a 3 × 2 × 2 factorial arrangement with three cultivars (AND 277, IAC-Milênio, and IAC-Carioca), two monosporic isolates (31-31 and 10-59), and two inoculum concentrations (2 × 104 and 4 × 104 conidia/mL). Disease severity was evaluated from 11 to 15 days after inoculation (d.a.i) using a diagrammatic scale and the Leaf Doctor. The results showed that 15 d.a.i., the concentration of the inoculum was no longer significant and that the isolates showed no significant difference in terms of pathogenicity. Moreover, in 15 d.a.i. it was possible to separate the cultivars into three phenotypic classes: resistant (AND-277), moderately susceptible (IAC-Milênio) and susceptible (IAC-Carioca). Considering the diagrammatic scale of notes, the cultivar AND 277 had a score of 2.3, while for the image evaluation, it was observed that only 0.65% of the total leaf area was affected by the disease. For this reason, 15 d.a.i. was recommended as the better day for evaluation, and the concentration of 2 × 104 conidia/mL may be adopted in order to reduce the amount of inoculum needed for the experiment. In addition, the evaluation with the Leaf Doctor app increases accuracy and improves discrimination between resistant and susceptible cultivars.   Key words: Pseudocercospora griseola, Phaseolus vulgaris, digital analysis, disease resistance.

Comparative Genomics Supports That Brazilian Bioethanol Saccharomyces cerevisiae Comprise a Unified Group of Domesticated Strains Related to Cachaça Spirit Yeasts.

Ethanol production from sugarcane is a key renewable fuel industry in Brazil. Major drivers of this alcoholic fermentation are Saccharomyces cerevisiae strains that originally were contaminants to the system and yet prevail in the industrial process. Here we present newly sequenced genomes (using Illumina short-read and PacBio long-read data) of two monosporic isolates (H3 and H4) of the S. cerevisiae PE-2, a predominant bioethanol strain in Brazil. The assembled genomes of H3 and H4, together with 42 draft genomes of sugarcane-fermenting (fuel ethanol plus cachaça) strains, were compared against those of the reference S288C and diverse S. cerevisiae. All genomes of bioethanol yeasts have amplified SNO2(3)/SNZ2(3) gene clusters for vitamin B1/B6 biosynthesis, and display ubiquitous presence of a particular family of SAM-dependent methyl transferases, rare in S. cerevisiae. Widespread amplifications of quinone oxidoreductases YCR102C/YLR460C/YNL134C, and the structural or punctual variations among aquaporins and components of the iron homeostasis system, likely represent adaptations to industrial fermentation. Interesting is the pervasive presence among the bioethanol/cachaça strains of a five-gene cluster (Region B) that is a known phylogenetic signature of European wine yeasts. Combining genomes of H3, H4, and 195 yeast strains, we comprehensively assessed whole-genome phylogeny of these taxa using an alignment-free approach. The 197-genome phylogeny substantiates that bioethanol yeasts are monophyletic and closely related to the cachaça and wine strains. Our results support the hypothesis that biofuel-producing yeasts in Brazil may have been co-opted from a pool of yeasts that were pre-adapted to alcoholic fermentation of sugarcane for the distillation of cachaça spirit, which historically is a much older industry than the large-scale fuel ethanol production.

POLYMORPHISM OF APPLE SCAB VENTURIA INAEQUALIS IN BELARUS

Study of Venturia inaequalis (Cooke) Wint. polymorphism causing apple scab is due to the need to elaborate and implement breeding programs allowing to develop highly resistant apple varieties and systems of phytosanitary protection against the pathogen. The objects of research were 125 V.inaequalis monoisolates isolated in pure culture from the affected apple leaves of 31 varieties of various genetic and geographical origins, growing in the collection orchard of the Institute for Fruit Growing. The study aimed to define the morphological and genetic structure of apple scab pathogen populations. Differences between scab isolates were identified by the growth pattern and a complex of morphological characters that made it possible to divide them into three morphotypes. The study of the morphocultural characters of scab isolates made it possible to determine the diversity of isolated strains that will be used to
 create artificial infectious backgrounds. The method of DNA fragment analysis of V.inaequalis monospore isolates using
 SSR markers allowed to identify high allelic polymorphism in the population of the apple scab causative agent — 218
 alleles were identified due to the presence of a sexual process in the pathogen’s life cycle. A high degree of genetic
 diversity of the apple scab causative agent was identified both within the population and a group of strains isolated
 from the same variety.

Mango malformation disease caused by Fusarium neocosmosporiellum in Mexico

Mango malformation disease caused by Fusarium spp. is one of the main limiting factors for mango production in Mexico. Fungal isolates obtained from symptomatic tissue from mango trees in Sinaloa, Mexico, were subjected to phylogenetic analysis of the TEF-1α gene, and evaluated for mating type, morphological markers and pathogenicity. Fifteen monosporic Fusarium isolates were identified as Fusarium neocosmosporiellum O’Donnell & Geiser. Phylogenetic analysis confirmed that the sequence obtained in this study corresponds to F. neocosmosporiellum. All 15 isolates were found to be pathogenic, but no differences were observed in aggressiveness among the isolates, which may be a result of low genetic variability within the fungal population. According to the morphological and molecular results, this study confirmed that the causal agent of mango malformation disease was F. neocosmosporiellum. This is the first report of mango malformation disease caused by this fungus in Mexico. This study provides a basis for the development and implementation of an integrated management system for this disease, confirms a new location of this fungus, and highlights that this pathogen is a potential threat to mango cultivation in Mexico.

First report of Penicillium olsonii causing postharvest fruit rot on tomato in Serbia.

Tomato (Solanum lycopersicum, L.) is one of the most important vegetable crop in Serbia, with a total production of 111,639 t in 2019 (Statistical Office of the Republic of Serbia). In July 2020, six tomatoes (cv. Balkan) with symptoms of fruit rot were collected from market in Belgrade, Serbia. The incidence of disease was about 2%, and the symptomatic samples were stored for 10 days after harvest. The initial symptoms on fruits were small circular, slightly sunken and water-soaked spots with white mycelia, that progressively expanded into larger grey lesions following the occurrence of sporulation. Isolations were conducted from one spot/fruit. Small pieces (2 to 3 mm2) from the margins of lesions were surface sterilized for 1 min in 1% NaOCl, washed twice with sterile distilled water, and cultivated on potato dextrose agar (PDA) at 25°C. The isolation frequency of Penicillium-like colonies was 100%. In total, six monosporic isolates were obtained and two isolates (SZ-20-6 and SZ-20-7) were selected as representative for morphological and molecular identification, and pathogenicity test. Morphological characteristics of both isolates were observed after growth on malt extract agar (MEA) for 7 days at 25ºC. On MEA, mycelia were white and colonies turned greyish-green with abundant sporulation. On the reverse sides colonies were pale yellow. The mean colony diameter on MEA for isolate SZ-20-6 was 25 ± 1.2 mm and 26 ± 1.0 mm for isolate SZ-20-7. The colony texture was velvety, without exudates and pigmentation. The conidiophores of both isolates were terverticillate, unbranched; phialides were flask shaped with a short neck, and conidia were smooth, greenish and subglobose to ellipsoidal. The conidial diameter for isolate SZ-20-6 was 3 to 4 × 2.5 to 3 µm, and for isolate SZ-20-7 was 3.5 to 4 × 2.5 to 3.5 µm (n =50). Based on these characteristics, isolates were identified as Penicillium olsonii (Pitt 1979). To confirm the morphological identification, genomic DNA was extracted from isolates (SZ-20-6 and SZ-20-7), and the rDNA ITS region and partial β-tubulin gene (BenA) were amplified using the primers ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. All sequences showed 99 to 100% similarity to P. olsonii and were deposited in GenBank (ITS, MW130235 and MW130236; BenA, MW145147 and MW145148). In multilocus phylogenetic analysis (ITS+BenA), isolates from this study clustered together with other P. olsonii sequences with 100% bootstrap support. To complete Koch's postulates, asymptomatic fruits of tomato cv. Balkan (five fruits per isolate) were superficially sterilized with 70% ethanol, wounded with a sterile needle and inoculated with 10 μl of a spore suspension (1 × 106 spores/ml). Five control fruits were inoculated with 10 μl of sterile distilled water. The experiment was repeated twice. After 7 days of incubation in a moisture chamber at 25°C, typical grey lesions developed on inoculated fruits. The control fruits remained symptomless. The isolates recovered from symptomatic fruits showed the same morphological features as the original isolates. P. olsonii was previously reported on tomato fruit only in Canada (Chatterton et al. 2012) and Pakistan (Anjum et al. 2018). To our knowledge, this is the first report of P. olsonii causing postharvest fruit rot on tomato in Serbia, and in Europe, as well. Therefore, it is essential to monitor spreading of P. olsonii on tomato and other crops in storages, and develop efficient disease management strategies.

<strong><em>Pseudopestalotiopsis gilvanii sp. nov.</em> and <em>Neopestalotiopsis formicarum</em> leaves spot pathogens from guarana plant: a new threat to global tropical hosts</strong>

Pestalotioid species (Pestalotiopsis, Pseudopestalotiopsis and Neopestalotiopsis) cause extremely damaging diseases in a wide range of hosts across the word. Recently, pestalotioid strains isolated from damaged guarana leaf tissue were subject to morphological and molecular characterization. Six monosporic isolates were obtained and analysed based on the following conidial characters: length, width, septation, absence or presence of basal appendage, number and length of apical appendages. For phylogenetic inference, sequences of the Internal Transcribed Spacer region (ITS), partial sequences of the genes encoding the translation elongation factor 1-α (tef1-α) and β-tubulin (tub2) were used. Three out of six strains analysed were identified as Neopestalotiopsis formicarum, while the three other isolates are described here as a new species of Pseudopestalotiopsis, named Ps. gilvanii sp. nov.. The pathogenicity of N. formicarum and Ps. gilvanii were confirmed following Koch’s postulate. Besides guarana, the potential of N. formicaram and Ps. gilvanii to cause diseases in other economically important tropical plants were investigated. Ps. gilvanii was pathogenic to açaí palms (Euterpe oleracea, E. precatoria), and oil palm (Elaeis guineensis), but not to banana (Musa paradisiaca var. pacovan) and rubber trees (Hevea brasiliensis). N. formicarum was not pathogenic to rubber trees but was pathogenic to other species tested. To our knowledge this is the first report of N. formicarum as a plant pathogen in the guarana plant, and Ps. gilvanii as novel plant pathogen capable of causing disease in important plant crops from tropical regions.