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

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

Monosporascus eutypoides, a Cause of Root Rot and Vine Decline in Tunisia, and Evidence that M. cannonballus and M. eutypoides Are Distinct Species.

Three Monosporascus eutypoides-like isolates recovered from cucurbit plants with symptoms of Monosporascus root rot and vine decline in Tunisia were compared to 28 isolates of M. cannonballus from 12 countries for phenotypic, genomic, and pathogenicity characteristics. Morphologically, M. cannonballus and M. eutypoides-like cultures were similar, each producing fertile perithecia in culture containing globose, smooth, dark brown to black ascospores. Nevertheless, all M. cannonballus isolates had one ascospore per ascus, while M. eutypoides-like isolates had mainly two to three ascospores per ascus (rarely one). The employment of the internal transcribed spacer (ITS) of nuclear ribosomal DNA, the elongation factor 1-α (EF-1α), and the ß-tubulin (ß-tub) gene sequence diversity analyses and the resulting phylogenies identified a level of polymorphism that enabled separation of M. cannonballus and M. eutypoides-like isolates. All isolates of M. cannonballus had identical EF-1α and ß-tub sequences irrespective of very diverse geographic origins, which were different from the EF-1α and ß-tub sequences of the M. eutypoides-like isolates (96 and 97% similarity, respectively). Similar results were obtained for the ITS region of rDNA. In addition, of three M. eutypoides-like isolates tested for pathogenicity, all three were pathogenic on watermelon, two were pathogenic on muskmelon, but only one was pathogenic on cucumber. The results demonstrate that the M. eutypoides-like isolates belong to the species M. eutypoides, and that M. cannonballus and M. eutypoides are distinct species.

First Report of Stem Rot of Papaya Caused by Fusarium solani Species Complex in Brazil.

In October 2010, 2-year-old papaya (cv. Hawaii) trees with high incidence of stem rot were observed during a survey conducted in Rio Grande do Norte state, northeastern Brazil. Stems showing reddish brown-to-dark brown symptoms were collected and small pieces (4 to 5 mm) of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with 0.5 g liter-1 streptomycin sulfate. Plates were incubated at 25°C with a 12-h photopheriod for 4 days. Pure cultures with white, fluffy aerial mycelia were obtained by subculturing hyphal tips onto PDA. Identification was made using morphological characteristics and DNA based molecular techniques. Colonies grown on PDA and Spezieller Nährstoffarmer agar (SNA) for 10 days at 25°C with a 12-h photoperiod were used for morphological identification (3). The fungus produced cream sporodochia and two types of spores: microconidia were thin-walled, hyaline, ovoid, one-celled, and 6.8 to 14.6 × 2.3 to 4.2 µm; macroconidia were thick walled, hyaline, slightly curved, 3- to 5-celled, and 25.8 to 53.1 × 3.9 to 5.7 µm. Fifty spores of each type were measured. Rounded, thick-walled chlamydospores were produced, with two to four arranged together. On the basis of morphological characteristics (1), three fungal isolates (CMM-3825, CMM-3826, and CMM-3827) were identified as Fusarium solani (Mart.) Sacc. and were deposited in the Culture Collection of Phytopathogenic Fungi of the Universidade Federal Rural de Pernambuco (Recife, Brazil). Single-spore isolates were obtained and genomic DNA of the isolates was extracted and a portion of the translation elongation factor 1-alpha (EF1-α) gene of the isolates was amplified and sequenced (2). When compared with sequences available in the GenBank and Fusarium-ID databases, DNA sequences of the three isolates shared 99 to 100% sequence identity with F. solani species complex (GenBank Accession Nos. JF740784.1, DQ247523.1, and DQ247017.1). Representative sequences of the isolates were deposited in GenBank (Accession Nos. JQ808499, JQ808500, and JQ808501). Pathogenicity tests were conducted with four isolates on 3-month-old papaya (cv. Hawaii) seedlings. Mycelial plugs taken from the margin of actively growing colonies (PDA) of each isolate were applied in shallow wounds (0.4 cm in diameter) on the stem (center) of each plant. Inoculation wounds were wrapped with Parafilm. Control seedlings received sterile PDA plugs. Inoculated and control seedlings (10 each) were kept in a greenhouse at 25 to 30°C. After 2 weeks, all inoculated seedlings showed reddish brown necrotic lesions in the stems. No symptoms were observed in the control plants. The pathogen was successfully reisolated from symptomatic plants to fulfill Koch's postulates. To our knowledge, this is the first report of F. solani species complex causing papaya stem rot in Brazil. Papaya is an important fruit crop in the northeastern Brazil and the occurrence of this disease needs to be taken into account in papaya production. References: (1) C. Booth. Fusarium Laboratory Guide to the Identification of the Major Species. CMI, Kew, England, 1977. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.