First Report of Cucurbita Blossom Blight and Fruit Rot Caused by Choanephora cucurbitarum in Mexico.

In December 2022, blossom blight, abortion, and soft rot of fruits were observed on Cucurbita pepo L. var. Zucchini in Mexico under greenhouse conditions (temperatures of 10 to 32°C and relative humidity up to 90%). The disease incidence in about 50 plants analyzed was around 70% with a severity of nearly 90%. Mycelial growth on flower petals and fruit rot with brown sporangiophores was observed. Ten disinfested fruit tissues in 1% NaClO for 5 min and then rinsed twice in distilled water from the lesion edges were placed on potato dextrose agar culture medium (PDA) supplemented with acid lactic and then, the morphological characterization was carried out in V8 agar medium. After 48 h of growth at 27°C, the colonies were pale yellow with diffuse cottony mycelia that were non-septate and hyaline and produced both sporangiophores bearing sporangiola and sporangia. The sporangiola were brown, ranged from ellipsoid to ovoid, and had longitudinal striations that measured 22.7 to 40.5 (29.8) µm x 16.08 to 21.9 (14.5) µm long and wide, respectively (n=100). The sporangia were subglobose, had a diameter of 127.2 to 281.09 (201.7) µm (n=50), and contained ovoid sporangiospores that measured 26.5 to 63.1 (46.7) µm x 20.07 to 34.7 (26.3) µm long and wide, respectively (n=100) which had hyaline appendages at the ends. Based on these characteristics, the fungus was identified as Choanephora cucurbitarum (Ji-Hyun et al. 2016). For molecular identification, DNA fragments for the internal transcribed spacer (ITS) and the large subunit rRNA 28S (LSU) regions for two representative strains (CCCFMx01 and CCCFMx02) were amplified and sequenced with the primer pairs ITS1-ITS4 and NL1-LR3 (White et al. 1990; Vilgalys and Hester 1990). The ITS and LSU sequences were deposited in GenBank database (Accession numbers OQ269823-24 and OQ269827-28, respectively) for both strains. The Blast alignment showed from 99.84 to 100% identity with Choanephora cucurbitarum strains JPC1 (MH041502, MH041504), CCUB1293 (MN897836), PLR2 (OL790293), and CBS 178.76 (JN206235, MT523842). To confirm the specie identification, the evolutionary analyses were conducted from the concatenated sequences of the ITS and LSU of C. cucurbitarum and other mucoralean species with the Maximum Likelihood method and Tamura-Nei model included in the software MEGA11. The pathogenicity test was demonstrated using five surface-sterilized zucchini fruits inoculated with a sporangiospores suspension containing a concentration of 1 x 105 esp/mL on two sites per fruit (20 µL each) that previously were wounded with a sterile needle. For fruit control, 20 µL of sterile water was used. Three days after inoculation under humidity conditions at 27°C, white mycelia and sporangiola growth with a soaked lesion were observed. That fruit damage was not observed on the control fruits. C.cucurbitarum was reisolated from lesions on PDA and V8 medium which was confirmed by morphological characterization fulfilling Koch's postulates. Blossom blight, abortion, and soft rot of fruits caused by C. cucurbitarum were observed on Cucurbita pepo and C. moschata in Slovenia and Sri Lanka (Zerjav and Schroers 2019; Emmanuel et al. 2021). This pathogen has the capability to infect a wide variety of plants worldwide (Kumar et al. 2022; Ryu et al. 2022). There are no reports of C. cucurbitarum causing agricultural losses in Mexico, and this is the first report causing the disease symptoms in Cucurbita pepo in this country; however, this fungus was found in the soil of papaya-producing areas and it is considered an important plant pathogenic fungus. Therefore, strategies for their control are highly recommended to avoid spreading the disease (Cruz-Lachica et al. 2018).

First Report of Ralstonia pseudosolanacearum Causing Wilt Disease in Tomato (Solanum lycopersicum L.) Plants from Mexico.

Mexico produces more than four million tons of tomato fruits and ranks tenth worldwide. In February 2022, tomato plants in a greenhouse in Culiacan, Sinaloa State, were affected by wilt diseases with an incidence of 20% and irreversible wilt and death of the infected plants (severity up 70%). When cut stems from affected plants, a reddish to brown discoloration of the vascular system was observed and these were disinfected with 1% NaClO for 5 min and then placed in a humid chamber. Characteristic milky-white exudate was obtained. From that exudate, irregular, mucoid, and white colonies with pink centres were obtained on casamino peptone glucose (CPG) plates supplemented with 1% 2,3,5-triphenyl 15 tetrazolium chloride (TZC); these characteristics are typical of the Ralstonia solanacearum species complex (RSSC) (Garcia et al., 2019). Identification of the pathogen was done by PCR using specific primer pairs reported by Paudel et al. (2022), RssC-wF3 (5'-TATATATCCTCGACTTTTCCATGAAGCTGTG-3') - RssCwR3 (5'-CTATATATATACCCCACTTGTTGAGGAACTG-3') and Rpseu-wF5 (5'-TTTTATTTTTTTGGTGTCCGGGCCAAGATAG-3') - Rpseu-wR5 (5'- TTATATTACTCGAACGTGCTGCAAAACCACT-3'), which amplified fragments of 162 and 251 bp for RSSC and Ralstonia pseudosolanacearum, respectively. Additionally, 759 (5'-GTCGCCGTCAACTCACTTTCC-3') - 760 (5'-GTCGCCGTCAGCAATGCGGAATCG-3') (Opina, et al., 1997) and Nmult21:1F (5'-CGTTGATGAGGCGCGCAATTT-3') - Nmult22:RR (5'- TCGCTTGACCCTATAACGAGTA-3') (Fegan and Prior, 2005) were used to generate 282 and 144 bp amplicons for RSSC and phylotype I, respectively. Subsequen to making the specific detection, the representative strain ClnMx was used to generate a sequence for the endoglucanase (egl) gene for separation into sequevars by using the primers Endo-F (5'- ATGCATGCCGCTGGTCGCCGC-3') and Endo-R (5'-GCGTTGCCCGGCACGAACACC-3'), which amplified a fragment of 750 bp (Fegan et al., 1998). The egl sequence (GenBank Access ON542479) showed 100% identity with the well-defined R. pseudosolanacearum sequevar 14, which was isolated from tomato plants from Senegal (UW763, I-14 GenBank Access CP051174) (Steidl et al., 2021), as well as, the strain MAFF 301070 (GenBank Access AB508612) from Japanese tomato. For pathogenicity tests, four 1-month-old tomato plants were infected using an insulin syringe that contained a pure bacterial suspension with approximately 2x108 CFU/mL. For each plant, 20 µL was infiltrated into the axil of the third upper leaf, and for untreated controls, tomato plants were infiltrated with sterile water. All plants were kept at 28°C under greenhouse conditions. Symptoms resembling those observed in the field were observed in inoculated plants six days after inoculation, and the plant pathogen was recovered on TZC medium. To confirm the bacteria identification a PCR using the specific primer pairs mentioned early was carried out. In contrast, water-treated control plants remained healthy. Koch's postulates were carried out twice with similar results. Ralstonia solanacearum species complex (RSSC) causes severe economic losses in many countries of the world because of their capability to infect a wide range of host plants, including potato, tomato, eggplant, tobacco, and, banana, among others. Ralstonia pseudosolanacearum has been reported to cause tomato wilt disease mainly on the Afro-Eurasian continent in areas such as Senegal, Cambodia, and Japan (Klass et al., 2019). To our knowledge, this is the first report of R. pseudosolanacearum causing bacterial wilt diseases in tomato plants from Mexico and because, the control of this bacteria is a challenge by the long survival time in soil, water, and infected plant tissues, the identification of this important pathogen could provide relevant information for developing management strategies.

First Report of Fusarium verticillioides Causing Cucumber Fruit Rot in Sinaloa, Mexico.

Cucumber (Cucumis sativus L.) is a fruit crop with high consumption worldwide. Mexico had a cucumber production of 826,485 tonnes in 2019. In December 2020, in a greenhouse in Sinaloa State, 18% of persian cucumber fruits with rot symptoms and the development of cottony white mycelia at both ends were observed similar to those described for Fusarium incarnatum (Garcia-Estrada et al., 2021). Isolation of the causal agent was carried out on PDA medium at 27°C for seven days from disinfested sections of cucumber tissues in NaOCl at 1% for one minute and then rinsed in distilled water. Morphological characterization was carried out on SNA medium, in which cultures was colorless and showed scarcely mycelia growth; however, microconidia were abundant and mainly showed clavate shaped measuring 16.6±2.2 x 5.32±1.0 µm (n=100). The morphological characteristics were similar to those described for Fusarium verticillioides (Nirenberhg, 1981). To confirm the species identity, the internal transcribed spacer (ITS) region and the actin (ACT), ß-tubulin (B-tub), calmodulin (CAL), and translation elongation factor 1-α (TEF1-α) genes were amplified and sequenced from one representative isolate: FPM03. These sequences were submitted to GenBank with the accession number MZ868200 for the ITS region and MZ955274 to MZ955277 for the ACT, B-tub, CAL, and TEF1-α regions. BLASTn analysis of the sequences showed 99 to 100% identity with several F. verticillioides sequence accession numbers MG515226, KU603765, MW402311, MW402449, and MW402113, which corresponded to strains CM1, CBS 576.78, and CBS 218.76. To evaluate Koch's postulates, ten healthy cucumber fruits were disinfected with 1% NaOCl for one min and then washed with distilled water. The fruits were inoculated with a single-spore suspension (3 × 104 conidia/mL) by spraying, as well as, five two-month-old cucumber plants. For controls, ten cucumber fruits and five plants were sprayed with sterile distilled water. All fruits were incubated in plastic bags at 25°C for four days and plants were placed under greenhouses conditions for a week. At 30 h after inoculation, all inoculated fruits showed soft rot symptoms at the fruit poles, and the development of white and cottony mycelia was observed at 48 h. Inoculated plants showed the symptoms mainly in the flower end of the fruits after three days. The symptoms observed under laboratory conditions were similar to those registered initially in the field. Samples of rotted tissues (fruit ends and flowers) from inoculated fruits were cultured on PDA medium; the resultant colonies showed similar characteristics to those obtained initially and the same pathogen was recovered. All control fruits and plants remained healthy, confirming pathogenicity. Fusarium verticillioides is primarily a maize pathogen causing stalk and ear rots globally, resulting in significant yield losses and reductions in grain quality; besides, this species produces large amounts of fumonisin B1 with high toxigenicity and is frequently found as food contaminant (Leslie and Sumerell, 2006). In addition, this pathogen was reported to affect sweet sorghum in Spain and banana in Jordan (Palmero et al., 2012; Salem et al., 2020). Recently, in Mexico, F. incarnatum was reported to cause soft rot in cucumber fruits (Garcia-Estrada et al., 2021); however, this is the first report of F. verticillioides affecting Mexican cucumber production. This information will be relevant for disease prevention and control.

Meloidogyne enterolobii egg extraction in NaOCl versus infectivity of inoculum on cucumber.

Extraction of eggs of Meloidogyne spp. in sodium hypochlorite (NaOCl) is a helpful procedure to assess the population levels and to obtain inoculum. In this sense, laboratory and greenhouse experiments were done to evaluate the effect of the NaOCl concentration on the viability of M. enterolobii eggs. Additionally, the objective of this investigation was to corroborate the preferable treatments to count populations in cucumber galled roots or to obtain inoculum of M. enterolobii. It was shown that the effect of the NaOCl concentration on the viability of M. enterolobii eggs is potentially detrimental. The NaOCl concentration caused a higher hatching, which in turn, resulted in non-infective larvae. Therefore, the best treatments to obtain inoculum of eggs of M. enterolobii included the 0.75% NaOCl (with 8-min stirring), 0.5% NaOCl (with stirring for 8, 12, and 16 min), and 0.3% NaOCl concentration (with stirring for 8, 12, 16, and 20 min). For a correct estimate of the egg population in roots, we show by several treatments that a concentration of 0.5% NaOCl (with stirring for 8, 12, and 16 min) and 0.75% NaOCl (with 8-min stirring) give the highest results.

First Report of Leaf Spot and Stem Canker on Watermelon Caused by Paramyrothecium foliicola in Mexico.

Watermelon is an important vegetable crop in Mexico and produced on 358,105 ha, with nearly 1.5 x 106 tons. In September 2019, brown, irregular shape to round lesions with concentric rings were observed on the leaves and stems of watermelon plants in Sonora State. The surface of the lesions contained abundant cup-shaped sporodochia covered by masses of olive-green to black conidia. Edge sections of symptomatic tissues were cut from the leaves, disinfected in 70% ethanol for 1 min and subsequently washed twice with distilled water. Disinfected tissue samples were transferred to PDA medium and incubated at 27°C for 15 days. White colonies were observed with spordochia arranged in concentric rings with characteristic of greenish-black masses of conidia. Spore masses stained with lactophenol blue were examined microscopically. Conidia were nonseptate and rod-shaped with rounded ends that measured 6.65 ± 0.54 x 1.56 ± 0.25 µm (n = 100). The characteristics of the fungus were similar to those reported for Paramyrothecium foliicola (Rennberger and Keinath, 2020). Molecular identification was performed on a representative isolate. RNA polymerase II second largest subunit (RPB2), calmodulin (CmdA) and the ß-tubulin (B-tub) genes were amplified and sequenced with the primer sets RPB2-5F2-RPB2-7cR, CAL228F-CAL737R and Bt2a-Bt2b, respectively. These sequences were submitted to GenBank with the acc. nos. MW116070 for RPB2, MW116071 for CmdA and MW116072 for B-tub. BLASTn analysis of the sequences demonstrated 99.34 to 100% identity with Paramyrothecium foliicola (acc. nos. MN398043, MN593713 and MN398138). Koch's postulates were verified on 15-day-old watermelon seedlings and mature fruit. One point of each of ten watermelon seedlings and six points of each of five fruit were marked for inoculation. A plug of mycelium obtained from a monosporic pure culture (grown for 15 days in PDA) was applied to each point without wounds. PDA only medium was included as the controls. The pathogenicity tests were repeated twice. Treated seedlings and fruit were kept in plastic bags at 27°C for 15 days. The first symptoms appeared 4 days after inoculation on the seedlings and 3 days after inoculation on the fruit. At the end of the test, the symptoms were similar to those observed initially in the field. The pathogen was re-isolated from lesion edges, and the morphological characteristics of the pathogen were determined to correspond with those of the inoculated fungus. Control seedlings and fruits remained healthy. P. foliicola has been reported to cause leaf spot disease on wild rocket and basil (Matic et al., 2019) and, recently, on watermelon in South Carolina (Rennberger and Keinath, 2020). To the best of our knowledge, this report is the first to describe P. foliicola causing leaf spot and stem canker on watermelon in Mexico.

First Report of Eggplant Fruit Rot Caused by Phytophthora nicotianae Breda de Haan in Mexico.

Eggplant (Solanum melongena L.) is an important solanaceous crop that is produced mainly in tropical and subtropical regions and is widely consumed worldwide. In 2018, eggplant production in Mexico was approximately 80000 t, and Sinaloa State contributed 96% of this production; however, this crop suffers significant losses from plant pathogens. In December 2019, fruits from commercial orchards (geographical coordinates: 24°45'39.39''N, 107°26'57.30''O) with visible brown soft rot and profuse white mycelia were analysed. On V8 medium, pieces of tissue obtained from the border of lesions were plated and incubated between 25°C and 36°C. After five days, a dense cottony mycelium with a slightly petaloid pattern was observed at 25°C and did not grow at 36°C. Isolates of that pathogen were heterothallic, and microscopic preparations showed development of coenocytic mycelium and spheroid sporangia that were noncaducous and papillate, measuring 35.6 ± 5.8 x 27.1 ± 4.4. Based on morphological characteristics, the eggplant soft rot causal agent was identified as Phytophthora nicotianae Breda de Haan (Erwin and Ribeiro, 1996). From a representative isolate denominated PhySm01, two DNA regions (internal transcribed spacer (ITS) and the large subunit ribosomal (28S)) were amplified and sequenced with ITS1-ITS4 and NL1-LR3 primers, respectively. The obtained ITS sequence (GenBank accession number MT508842) showed 100% identity with several P. nicotianae sequences (Access MT065840, MH290435 and MG570057) with 100% query coverage and 740 matching nucleotides. For the 28S sequence (accession number MT508843), the identity with strains N° Access EU080889 and EU080508 of P. nicotianae was 99.86%, with 100 query coverage and 729 matching nucleotides. Further, phylogenetic analysis from P. nicotianae strain PhySm01 and GenBank reference sequences was carried out by Maximum Likelihood method with Mega 7 software based on the ITS sequences, which verified the species identification. To fulfill Koch's postulates, a suspension containing 1 x 104 zoospores/mL of the oomycete isolated from the original diseased eggplant fruit was used to inoculate ten healthy and disinfested fruits. Sterile water was used as a control. Three wounds per fruit were made with a sterile needle, and 20 µL of the zoospore suspension (or water) was placed on each. All fruits were placed into plastic bags with moistened paper and incubated at 25°C for three days. Thisest was repeated twice with similar results. Initial symptoms developed 24 hours after inoculation with brown soft tissue forming around the inoculated area, and profuse soft rot accompanied by white mycelium was observed two days after inoculation. No symptoms developed on the control fruits during this time. Pieces of necrotic tissue were plated on V8 medium and incubated as described previously. The reisolated pathogen was compared to the original isolate and had the same morphological characteristics. Phytophthora nicotianae has a worldwide distribution and can infect multiple solanaceous crops, including tobacco (Gallup et al., 2018). In addition, it has been reported on other hosts outside of the Solanaceae family, causing economically important losses in citrus and strawberry. In India, it causes necrosis of citrus fruits and roots, leading to tree decline (Das et al., 2016), and in the United States, it causes crown rot of strawberry (Marin et al., 2018). In Mexico, P. nicotianae has been reported on vinca (Alvarez-Rodriguez et al., 2013); however, this is the first report of P. nicotianae causing fruit soft rot of eggplant, and this pathogen represents a new threat when the environment is favourable for disease outbreaks. References Alvarez-Rodriguez, B. et al. 2013. Plant Dis. 97: 1257. https://doi.org/10.1094/PDIS-04-13-0400-PDN Das, A. K. et al. 2016. J. Plant Pathol. 98: 55. DOI: 10.4454/JPP.V98I1.038 Erwin, D. C. and Ribeiro, O. K. 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN. 562 Pp. Gallup, C. A. et al. 2018. Plan Dis. 102: 1108. https://doi.org/10.1094/PDIS-02-17-0295-RE Marin, M. V. et al. 2018. Plant Dis. 102:1463. https://doi.org/10.1094/PDIS-08-17-1333-PDN.