First report of Lasiodiplodia theobromae causing leaf rot on Agave angustifolia in Mexico.

The agave crop (Agave angustifolia), is of economic importance for Mexico, for the agave is made mainly an alcoholic beverage called locally mezcal. In the state of Guerrero, in the municipality of Huitzuco de los Figueroa (18.2510026N, 99.2320182W, 1196 m above sea level), a severe disease affecting agave leaves was detected. The field symptoms consisted of pale to brown dark descending lesions, covering >50% of the leaf surface, in which the presence of pycnidia was observed. In an estimated area of 0.5 ha, the estimated incidence was 67% (n=100 plants). Symptomatic fragments from leaves (approximately 0.5 cm) were taken, superficially disinfected with 1% NaClO, and rinsed twice with sterile distilled water. Then they were transferred to potato dextrose agar (PDA) medium, and incubated at 28 °C. After five days, twelve representative isolates were selected and purified by the hyphal tip technique. In the PDA medium, the colonies were initially light gray, later they became dark, and after 22 days of incubation, the development of numerous dark pycnidia was observed on the surface of the medium. Initially, immature hyaline conidia, unicellular, oval, and double-walled were observed. The mature conidia were dark brown, oval, with one septum and longitudinal striation, and measured 17.5 to 27 [average 25.3 µm; n=50] × 10.5 to 15.7 [average 13.9 µm; n=50]. Based on the morphological characteristics, the fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Alves et al. 2008). Isolates LAS3 and LAS4 were used for molecular identification, this was done by amplifying the regio internal transcribed spacer (ITS) of rDNA with primers ITS1 and ITS4 (White et al. 1990) and translation elongation factor 1-alpha ( EF-1α) genes using primers EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting sequences were deposited in GenBank (LAS3; ON391564 and LAS4; ON391565 for ITS, and LAS3; ON368190 and LAS4; ON368191 for EF-1α). BLASTn analysis sequences of isolated LAS3 and LAS4 revealed for ITS 98.6% identity with L. theobromae (MK934699.1), and for EF-1α indicated 100% identity (MF422024.1). From concatenated sequences ITS-EF-1α regions, a phylogenetic analysis was carried out in MEGA X software, using the Maximum Likelihood and Kimura 2-parameter model with 1,000 bootstraps replicated; isolates LAS3 and LAS4 were clustered in the clade of the members of L. theobromae strains CAA006 (Alves et al. 2006), and INTA-IMC 1601 (Perez et al. 2018). The pathogenicity tests were carried out on 10 healthy 3 year-old agave plants, in which the mycelium of the LAS4 isolate was inserted at three equidistant points/leaf, using a sterile toothpick. Five healthy agave plants were inoculated only with sterile PDA as control treatment. The inoculated plants were covered with transparent plastic bags and housed in a greenhouse at 28 °C. After seven days, similar symptoms to those observed in the field were observed in all inoculated plants. Control plants did not develop symptoms. The fungus L. theobromae was re-isolated again from the infected leaves, fulfilling Koch's postulates. In China, L. theobromae has been reported as the cause of leaf rot on A. sisalana (Xie et al. 2016). To our knowledge, this is the first report of L. theobromae causing leaf rot on A. angustifolia in Mexico. This research is useful to design management strategies for leaf rot disease for local farmers of A. angustifolia.

First Report of Bud Soft Rot on Agave angustifolia Caused by Pantoea dispersa in Mexico.

The agave (Agave spp.) is an important crop in México, with 120,897 ha grown mainly for alcoholic beverage production (SIAP, 2019). In September 2020, in the municipality of Huitzuco de los Figueroa (18.328692 N; 99.3998 W), Guerrero State, México, a serious disease was observed affecting Agave angustifolia. Disease incidence was 8% of 150 plants sampled over an approximate area of 2.5 ha. Initial symptoms of soft rot of the bud developed and produced an abundant exudate which appeared from the apical part to the base of the plant. In severe infections, the plants showed total maceration of the bud, and consequently death of the plants was observed. Symptomatic plant tissue was superficially disinfected with 1% NaOCl for 30 s, and rinsed in sterile water three times. The disinfected tissues were macerated and with a loop spread in Nutrient Agar. The plates were incubated at 28 ° C for 2 days. Yellowish bacterial colonies were isolated, and eight colonies were selected for characterization. The bacterial strains were gram negative and rod-shaped, negative for fluorescent pigment tests and Kovacs' oxidase. Two isolates designated AGA1 and AGA2 were identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F / 1492R (Lane 1991), and partial fusA, rpoB, and gyrB genes (Delétoile et al. 2009). Sequences were deposited in GenBank, with the accession numbers for 16S rRNA, AGA1 as MW548406 and AGA2 as MW548407; for specific genes fusA (AGA1 = MW558445, AGA2 = MW558446), rpoB (AGA1 = MW558447, AGA2 = MW558448) and gyrB (AGA1 = MW558449, AGA2 = MW558450), and they were compared with the sequences available in GenBank using BLASTn. 16S rRNA gene sequences for AGA1 and AGA2 aligned with Pantoea dispersa (MT921704.1, 99.9% identity). Housekeeping genes also aligned 99 to 100% to P. dispersa (fusA = 100%, CP045216.1; rpoB = 99.8% MH015167.1 and gyrB = 99%, MK928270.1). Phylogenetic analysis of concatenated genes showed that strains AGA1 and AGA2 cluster with P. dispersa. To confirm pathogenicity, eight plants of six-month-old A. angustifolia were inoculated with strain AGA1 using sterile toothpicks dipped in 108 CFU/ml bacterial suspension. The toothpicks were inserted in the middle part of the bud. Four plants were inoculated with sterile water as control. The plants were covered with plastic bags and housed in a greenhouse (average temperature and relative humidity of 25 ° C and 85%, respectively). Pathogenicity tests were repeated two times. After seven days, all inoculated plants developed symptoms similar to those observed in the field. Control plants did not show symptoms. From the plants that showed symptoms, the pathogen was reisolated again and was identified by morphological and molecular characterization, following the method previously described, fulfilling Koch's postulates. In México, Erwinia cacticida and Pantoea ananatis has been previously reported on A. tequilana that as causing soft rot and red leaf ring, respectively (Jimenez-Hidalgo et al. 2004; Fucikovsky and Aranda 2006). To our knowledge, this is the first report of P. dispersa causing bud soft rot on A. angustifolia in México. More studies monitoring and control strategies of bud soft rot on A. angustifolia are required.

[Spatial distribution of anthracnose (Colletotrichum gloeosporioides Penz) in avocado in the State of Mexico, Mexico]. / Distribución espacial de antracnosis (Colletotrichum gloeosporioides Penz) en aguacate en el Estado de México, México.

Persea americana is a species of great nutritional and economic importance for Mexico, however, like any other agricultural crop, it is affected by pests and diseases that limit its worldwide commercialization. The phytopathogenic fungus Colletotrichum gloeosporioides is the causative agent of anthracnose in avocado and manifests itself in the early stages of fruit development as well as in post-harvest and storage, under conditions of high relative humidity (80%) and at temperatures from 20°C, causing losses economic up to 20% of production. Applying geostatistical methods the present study aims to define the spatial distribution of anthracnose in Hass avocado fruits in four municipalities of the State of Mexico during the period from January to June 2017. The results show that the distribution of anthracnose was adjusted to gaussian and exponential models in most, the infestation maps made through the kriging show more than one center of aggregation of the disease, based on it the infested surface was estimated, finding an infestation of more than 50% in the first samples and up to 98% in the samplings belonging to the month of June in all the areas studied.

Distribution and host range of Bephratelloides cubensis Ashmead (Hymenoptera: Eurytomidae) in Mexico.

The annona fruit borer wasp Bephratelloides cubensis Ashmead was recorded for the first time damaging "ilama" fruits Annona diversifolia (Anonaceae) in Coatlan del rio, Morelos, Cacahuamilpa and El Ocotillo, Guerrero, Mexico. Geographical distribution, hosts and damage are mentioned.

[Persea schiedeana (Lauraceae), a new host of Heilipus lauri Boheman (Coleoptera: Curculionidae) in Veracruz, Mexico]. / Persea schiedeana (Lauraceae), nuevo hospedero de Heilipus lauri Boheman (Coleoptera: Curculionidae) en Veracruz, México.

The presence of the Molytinae Heilipus lauri Boheman is reported for the first time attacking fruits of Persea schiedeana, commonly called 'chinene' in Huatusco and Zongolica, state of Veracruz, Mexico.

[Genitalia of three species of Heilipus Germar (Coleoptera: Curculionidae) that damage avocado fruits (Persea americana Mill.) in Mexico and Costa Rica]. / Genitalia de tres especies de Heilipus Germar (Coleoptera: Curculionidae) que dañan frutos de aguacate (Persea americana Mill) en México y Costa Rica.

The male and female genitaliae of three species of the genus Heilipus Germar (H. lauri Boheman, H. pittieri Barber and H. trifasciatus Fabricius) that damage avocado fruits (Persea americana Mill.) in Mexico and Costa Rica are described and illustrated. The aedeagus, spiculum gastrale, styli of 8th sternite are different in each one of the three species studied and can be used for specific identification.

Distribution and host range of Bephratelloides cubensis Ashmead (Hymenoptera: Eurytomidae) in Mexico

The annona fruit borer wasp Bephratelloides cubensis Ashmead was recorded for the first time damaging "ilama" fruits Annona diversifolia (Anonaceae) in Coatlan del rio, Morelos, Cacahuamilpa and El Ocotillo, Guerrero, Mexico. Geographical distribution, hosts and damage are mentioned.

Persea schiedeana (Lauraceae), nuevo hospedero de Heilipus lauri Boheman (Coleoptera: Curculionidae) en Veracruz, México / Persea schiedeana (Lauraceae), a new host of Heilipus lauri Boheman (Coleoptera: Curculionidae) in Veracruz, Mexico

The presence of the Molytinae Heilipus lauri Boheman is reported for the first time attacking fruits of Persea schiedeana, commonly called "chinene" in Huatusco and Zongolica, state of Veracruz, Mexico.

Se reporta por primera vez la presencia del Molytinae Heilipus lauri Boheman atacando frutos de Persea schiedeana, comúnmente conocidos como "chinene" in Hustusco y Zongolica, Veracruz, México.

Genitalia de tres especies de Heilipus Germar (Coleoptera: Curculionidae) que dañan frutos de aguacate (Persea americana Mill) en México y Costa Rica / Genitalia of three species of Heilipus Germar (Coleoptera: Curculionidae) that damage avocado fruits (Persea americana Mill.) in Mexico and Costa Rica

The male and female genitaliae of three species of the genus Heilipus Germar (H. lauri Boheman, H. pittieri Barber and H. trifasciatus Fabricius) that damage avocado fruits (Persea americana Mill.) in Mexico and Costa Rica are described and illustrated. The aedeagus, spiculum gastrale, styli of 8th sternite are different in each one of the three species studied and can be used for specific identification.

La genitalia masculina y femenina de tres especies de Heilipus Germar (H. lauri Boheman, H. pittieri Barber y H. trifasciatus) que dañan frutos de aguacate (Persea americana Mill.) en México y Costa Rica es descrita e ilustrada. El edeago, espícula gastrale, y estilos del esternitos VIII son diferentes en cada una de las tres especies estudiadas y pueden ser usadas para la identificación específica.