Seedborne fungi and viruses in bean crops (Phaseolus vulgaris L.) in Nicaragua and Tanzania

The common bean (Phaseolus vulgaris L.) is an important legume crop grown widely around the world due to its high nutritional values. In developing countries of Africa and Latin America (e.g. Tanzania and Nicaragua) bean crop is linked to food security and income generation especially in poorest groups made up of small farmers. Disease problems, pests, unimproved seeds, inappropriate agricultural management and environmental conditions are often the main constraints in bean crop production. In Nicaragua and Tanzania, the unavailability of certified seed of local bean varieties causes significant losses in yield and quality. Other limitation present in Nicaragua and Tanzania concerning bean diseases is that seed-borne fungi and viruses has gained little attention. Thus, for better understanding in seed-borne fungi and viruses associated with beans we investigated seed-borne fungi in an important new local bean variety. Sampling from four seed storehouses and six seed lots of cv. INTA Rojo was done in the main bean production areas in Nicaragua. In addition, to detect viruses infecting bean plants, we surveyed seedborne viruses in landraces and new common bean varieties introduced to Nicaragua (Central America) as well as improved varieties grown in Tanzania (eastern Africa). The main results for seedborne fungi included 87 pathogenic isolates from eight genera: Fusarium spp. (F. chlamydosporum, F. equiseti, F. incarnatum), Lasiodiplodia theobromae, Macrophomina phaseolina, Corynespora cassiicola, Colletotrichum capsici, Colletotrichum gloeosporioides, Diaporthe sp. (Phomopsis), Aspergillus flavus, and Penicillium citrinum. Subsequently, results based on germination in seed lots of common bean (‘INTA Rojo’) from four bean production areas in Nicaragua showed that germination was constantly less than 40% and could be as low as 16%, indicating disastrous yield losses for producers. Four different species-specific primer pairs to detect F. equiseti, F. chlamydosporum, F. incarnatum, C. capsici, C. gloesporiodes and C. cassiicola were developed in this study based on sequence alignment of the internal transcribed spacer sequences (ITS1 and ITS2) from different fungi. They were tested in pure cultures of fungi and used successfully in detection of fungal pathogens from infected plants. These specific primers are able to give the basis to be used in seed health inspection (seeds and plants) for further research of the epidemiology, ecology, and control of the pathogenic fungi of common beans in the field. The presence of Phaseolus vulgaris endornavirus 1 (PvEV-1) and PvEV-2 was detected in Nicaraguan and Tanzanian bean varieties. Likewise, Cowpea mild mosaic virus was detected in one region of Tanzania. There is apparently indication that the new Nicaragua bean varieties are carrying virus resistance genes because no seedborne viruses were found in them. In Tanzania improved protection against pathogenic seed-borne viruses is developed by resistance breeding. These findings are the first report showing that several pathogenic seed-borne fungi occur in Nicaraguan beans. Previously, little information has been available on pathogenic fungi such as F. equiseti, F. incarnatum, L. theobromae, C. cassiicola, and Diaporthe spp in Nicaraguan common beans. This study has contributed in taking first steps to improve the pathological and genetic components in the national seed production system in Nicaragua and Tanzania by providing new knowledge concerning seed-borne pathogens associated with common bean.Tarhapapu (Phaseolus vulgaris L.) on tärkeä palkokakasvi, jota viljellään laajalti ympäri maailmaa korkean ravintoarvonsa vuoksi. Afrikan ja Latinalaisen Amerikan kehitysmaissa (esim. Tansaniassa ja Nicaraguassa) pavuntuotanto on tärkeää ruokaturvan ja tulonmuodostuksen näkökulmasta etenkin köyhimmissä väestöryhmissä, jotka koostuvat pienviljelijöistä. Kasvitautiongelmat, tuholaiset, terveiden siementen puute, viljelymenetelmien kehittymättömyys ja ympäristöolot ovat usein keskeisinä rajoituksina pavuntuotannolle. Nicaraguassa ja Tansaniassa paikallisten papulajikkeiden sertifioitujen siementen puute aiheuttaa merkittäviä sadon määrän ja laadun menetyksiä. Nicaraguassa ja Tansaniassa viljelyä rajoittaa se, että siemenpavuissa leviävät sienet ja virukset ovat jääneet vähälle huomiolle. Tässä tutkimuksessa tunnistettiin ja eristettiin pavunsiementen sieniä tärkeässä, uudessa papulajikkeessa INTA Rojo. Näytteenotto tehtiin neljästä siemenvarastosta ja kuudesta siemenerästä Nicaraguan papujen päätuotantoalueilla. Viruksia tutkittiin myös maatiaispapulajikkeista ja äskettäin viljelyyn otetuista uusista papulajikkeista Nicaraguassa ja Tansaniassa. Siemenpavuista eristetyistä sienistä 87 sienikantaa aiheutti taudinoireita pavuissa. Ne kuuluivat kahdeksaan sukuun: Fusarium spp. (F. chlamydosporum, F. equiseti, F. incarnatum), Lasiodiplodia theobromae, Macrophomina phaseolina, Corynespora cassiicola, Colletotrichum capsici, Colletotrichum gloeosporioides, Diaporthe sp. (Phomopsis), Aspergillus flavus, ja Penicillium citrinum. Nicaraguassa neljältä viljelyalueelta tutkittujen papujen (INTA Rojo) itävyys oli huono, 16-40%, mikä viittaa sienten merkittävään satotasoa vähentävään vaikutukseen, kun tervettä siementä ei ole saatavilla. Tässä tutkimuksessa kehitettiin neljä erilaista lajispesifistä alukeparia sienten F. equiseti, F. chlamydosporum, F. incarnatum, C. capsici, C. gloesporiodes ja C. cassiicola havaitsemiseksi PCR-menetelmällä (polymerase chain reaction). Testi perustuu ribosomaalista RNA:ta tuottavien geenien välialueisiin (ITS1 ja ITS2), joiden DNA-sekvenssi vaihtelee sienilajien välillä. Phaseolus vulgaris endornavirus 1:n (PvEV-1) ja PvEV-2:n läsnäolo havaittiin pavuissa sekä Nicaraguassa että Tansaniassa. Samoin Cowpea mosaic virus havaittiin pavuissa yhdellä Tansanian alueella. On ilmeistä, että uudet Nicaraguassa viljeltävät papulajikkeet ovat aiempaa kestävämpiä viruksille, koska viruksia ei näistä papulajikkeista löytynyt. Tansaniassakin papujen viruskestävyyttää on kehitetty kestävyysjalostuksella. Tämän tutkimuksen tulokset edustavat ensimmäistä laajempaa analyysiä siemenpavuissa esiintyvistä patogeenisista sienistä Nicaraguassa ja tuo esiin sienten, kuten F. equiseti, F. incarnatum, L. theobromae, C. cassiicola ja Diaporthe-lajien merkityksen siementen itävyyden ja siten satotason heikentäjinä. Tutkimuksen tulokset edesauttavat Nicaraguan ja Tansanian kansallisen siementuotantojärjestelmän parantamista antamalla uutta tietoa siemenpapujen mukana leviävistä ja satotasoa heikentävistä taudinaiheuttajista

Seedborne Pathogenic Fungi in Common Bean (Phaseolus vulgaris cv. INTA Rojo) in Nicaragua

Common bean (Phaseolus vulgaris L.) is an important legume with high nutritional value. In Nicaragua, certified healthy seeds of local bean varieties are not available, and seedborne fungi have gained little attention. Here, were surveyed seedborne pathogenic fungi in an important local bean cultivar, `INTA Rojo'. Beans grown in the four main production areas in Nicaragua (Boaco, Carazo, EstelõÂ, Matagalpa) for future use as seed stock were sampled from four seed storehouses and six seed lots. A total of 133 fungal strains were isolated from surface-sterilized beans and inoculated to healthy lima beans (Phaseolus lunatus) under controlled conditions. Eighty-seven isolates caused symptoms of varying severity in the seedlings, including discoloration, necrotic lesions, cankers, rot, and lethal necrosis. Pathogenic isolates were divided into eight phenotypically distinguishable groups based on morphology and growth characteristics on artificial growth medium, and further identified By analysis of the internal transcribed spacer sequences (ITS1 and ITS2) of the ribosomal RNA genes. The pathogenic isolates belonged to eight genera. Fusarium spp. (F. chlamydosporum, F. equiseti, F. incarnatum), Lasiodiplodia theobromae, Macrophomina phaseolina, and Penicillium citrinum were the most damaging and common fungi found in the seed lots. Furthermore, Corynespora cassiicola, Colletotrichum capsisi, Colletotrichum gloeosporioides, Aspergillus flavus, and Diaporthe sp. (Phomopsis) were seedborne in cultivar `INTA Rojo' and found to be pathogenic to bean seedlings. This study reveals, for the first time, many seedborne pathogenic fungi in beans in Nicaragua; furthermore, prior to this study, little information was available concerning F. equiseti, F. incarnatum, L. theobromae, C. cassiicola, and Diaporthe spp. as seedborne pathogens of common bean. Our results lay the basis for developing diagnostic tools for seed health inspection and for further study of the epidemiology, ecology, and control of the pathogenic fungi of common beans in the field.Common bean (Phaseolus vulgaris L.) is an important legume with high nutritional value. In Nicaragua, certified healthy seeds of local bean varieties are not available, and seedborne fungi have gained little attention. Here, were surveyed seedborne pathogenic fungi in an important local bean cultivar, `INTA Rojo'. Beans grown in the four main production areas in Nicaragua (Boaco, Carazo, Estelf, Matagalpa) for future use as seed stock were sampled from four seed storehouses and six seed lots. A total of 133 fungal strains were isolated from surface-sterilized beans and inoculated to healthy lima beans (Phaseolus lunatus) under controlled conditions. Eighty-seven isolates caused symptoms of varying severity in the seedlings, including discoloration, necrotic lesions, cankers, rot, and lethal necrosis. Pathogenic isolates were divided into eight phenotypically distinguishable groups based on morphology and growth characteristics on artificial growth medium, and further identified by analysis of the internal transcribed spacer sequences (ITS1 and ITS2) of the ribosomal RNA genes. The pathogenic isolates belonged to eight genera. Fusarium spp. (F. chlamydosporum, F. equiseti, F. incarnatum), Lasiodiplodia theobromae, Macrophomina phaseolina, and Penicillium citrinum were the most damaging and common fungi found in the seed lots. Furthermore, Corynespora cassiicola, Colletotrichum capsisi, Colletotrichum gloeosporioides, Aspergillus flavus, and Diaporthe sp. (Phomopsis) were seedborne in cultivar `INTA Rojo' and found to be pathogenic to bean seedlings. This study reveals, for the first time, many seedborne pathogenic fungi in beans in Nicaragua; furthermore, prior to this study, little information was available concerning F. equiseti, F. incarnatum, L. theobromae, C. cassiicola, and Diaporthespp. as seedborne pathogens of common bean. Our results lay the basis for developing diagnostic tools for seed health inspection and for further study of the epidemiology, ecology, and control of the pathogenic fungi of common beans in the field.Peer reviewe

Pathogenic seedborne viruses are rare but Phaseolus vulgaris endornaviruses are common in bean varieties grown in Nicaragua and Tanzania

Common bean (Phaseolus vulgaris) is an annual grain legume that was domesticated in Mesoamerica (Central America) and the Andes. It is currently grown widely also on other continents including Africa. We surveyed seedborne viruses in new common bean varieties introduced to Nicaragua (Central America) and in landraces and improved varieties grown in Tanzania (eastern Africa). Bean seeds, harvested from Nicaragua and Tanzania, were grown in insect-controlled greenhouse or screenhouse, respectively, to obtain leaf material for virus testing. Equal amounts of total RNA from different samples were pooled (30-36 samples per pool), and small RNAs were deep-sequenced (Illumina). Assembly of the reads (21-24 nt) to contiguous sequences and searches for homologous viral sequences in data-bases revealed Phaseolus vulgaris endornavirus 1 (PvEV-1) and PvEV-2 in the bean varieties in Nicaragua and Tanzania. These viruses are not known to cause symptoms in common bean and are considered non-pathogenic. The small-RNA reads from each pool of samples were mapped to the previously characterized complete PvEV-1 and PvEV-2 sequences (genome lengths ca. 14 kb and 15 kb, respectively). Coverage of the viral genomes was 87.9-99.9%, depending on the pool. Coverage per nucleotide ranged from 5 to 471, confirming virus identification. PvEV-1 and PvEV-2 are known to occur in Phaseolus spp. in Central America, but there is little previous information about their occurrence in Nicaragua, and no information about occurrence in Africa. Aside from Cowpea mild mosaic virus detected in bean plants grown from been seeds harvested from one region in Tanzania, no other pathogenic seedborne viruses were detected. The low incidence of infections caused by pathogenic viruses transmitted via bean seeds may be attributable to new, virus-resistant CB varieties released by breeding programs in Nicaragua and Tanzania.Peer reviewe

Caracterización biológica y molecular de geminivirus que afectan el cultivo del tomate (Lycopersicon esculentum Mill) en Nicaragua

El objetivo del presente trabajo consistió en la caracterización biológica y molecular de geminivirus que afectan el cultivo del tomate (Licopersicon esculentum Mili.), el estudio se realizó en la Universidad Nacional Agraria (UNA), en el período de enero 2001 a enero 2002. Se efectuó en tres fases: la primera fase de invernadero, donde se utilizaron plantas de tomate de la variedad UC-82, crías de mosca blanca (Bemisia tabaci Genn), fuente original del aislado del geminivirus de Condega, con el objetivo de caracterizar biológicamente el geminivirus, a través de periodos de adquisición, inoculación y retención, previamente para la realización de estos periodos, se determinó el porcentaje de transmisibilidad de la colonia de mosca blanca, el cual fue de 45% Jo que indicó que para asegurar la transmisibilidad del virus fue necesario usar 5 moscas por planta. Los períodos de adquisición e inoculación fueron los tiempos de 2, 5, 10 y 30 minutos, 1, 2, 4, 8, 10, 12, 18, 24 y 48 horas con 3 repeticiones de cada periodo, los datos obtenidos de estos periodos fueron analizados mediante un análisis de regresión logístico binario, resultando que el vector fue capaz de adquirir y transmitir el virus tanto en 1 O minutos (40% y 33%) como en 48 horas (60% y 66.66%) respectivamente, respecto al periodo de retención se estableció realizándose un promedio de los porcentajes de severidad de las 3 repeticiones mostrando como resultado que el vector conservó su capacidad de transmisión del virus hasta el sexto día con 45.07% de transmisibilidad, también se evaluaron 5 especies de la familia solanáceas injertándolas con plantas afectadas con el genminivirus de Condega, para conocer cuáles son hospederas del virus, resultando la especie Nicotiana tabacum cv. Benthamiana con síntomas del virus. En esta misma fase se evaluaron 4 materiales de tomate (55, 111, 148, 112) usando como testigo la variedad UC-82, se utilizó un Diseño Completamente al Azar (DCA) y se evaluó la variable severidad de la enfermedad a los datos obtenidos a través de ANDEVA y separación de medias según Duncan, estos materiales se evaluaron mediante cuatro tratamientos (inoculaciones), el primero a los 7 ddt, el segundo a los 14 ddt, el tercero a los 21 ddt y el último tratamiento fueron las plantas sin inocular (testigos), el análisis indicó que la variedad UC-82 fue la más susceptible ante el complejo mosca blanca-geminivirus presentando mayores porcentajes de severidad y el material 112 resultó el más resistente, con menos porcentajes de severidad. La segunda fase se realizó fuera del invernadero, evaluándose ante el complejo mosca blanca-geminivirus 3 materiales, incluyendo la variedad UC-82 como testigo. Se utilizó un Diseño completamente al Azar, tomando en cuenta las variables severidad, incidencia y población de mosca blanca, A los datos obtenidos se les realizó ANDEVA y separación de medias según Duncan, el que mostró que el material 55 fue el mejor, ya que presentó menos porcentaje de severidad (40%), el peor material fue lavar. UC-82 ya que mostró 58.75% de severidad, con relación a la incidencia todos los materiales evaluados resultaron al final de los muestreos con igual porcentaje de incidencia (!00%) y en el caso de la población de mosca blanca la variedad UC-82 fue la que presentó más adultos por planta en comparación con el resto de materiales. La tercera fase fue realizada en el laboratorio de biología molecular en la UNA y Suecia con el propósito de caracteri7ar molecularmente los geminivirus de Condega., Santa Lucía y Sébaco, secuenciando un fragmento de 1200 pares de bases del componente A para comparar a través de las secuencias de nucleótidos los porcentajes de similitud que existen entre ellos, dando como resultado que el geminivirus de Sébaco con el de Santa Lucía alcanzó mayor porcentaje de similitud con un 95.2 con respecto al geminivirus de Condega que obtuvo un 89.0%

Emergence of beans (cv. INTA Rojo) and the portion of emerged seedlings showing disease-like symptoms 15 days after planting under controlled conditions.

<p>Six bean storehouses belonging to different small farmers’ cooperatives were sampled in four regions in Nicaragua. Eight subsamples (50 seeds each) were taken from each store and planted under controlled conditions. Least significant difference of means for emergence = 10.4 (p = 0.00006; Tukey, α = 0.05).</p

Symptoms of lima bean seedlings caused by fungal isolates of phenogroup IV.

<p>(a) to (c), inoculated plants at 20 dpi, and (d), non-inoculated control. Necrosis in the middle of the stem corresponds to the position of inoculum placed in the test tube. All inoculated seedlings display severe apical necrosis.</p

Phylogenetic grouping of the fungal isolates in phenogroup V.

<p>Partial ITS1 and the whole 5.8S and ITS2 sequences (~450 nt) of the fungi isolated from beans grown in Boaco (bold letters) were included in the phylogenetic analysis with sequences of <i>Colletotrichum capsici</i> (clade 1), <i>C</i>. <i>gloeosporioides</i> (clade 3), <i>C</i>. <i>parsonsiae</i> (JQ005233), <i>C</i>. <i>petchii</i> (JQ005223), and <i>C</i>. <i>constrictum</i> (JQ00538) (clade 2) obtained from sequence databases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168662#pone.0168662.s001" target="_blank">S1 Table</a>). Numbers at branches represent bootstrap values of 1000 replicates. Only bootstrap values of ≥70% are shown. Scale indicates Kimura units in nucleotide substitutions per site.</p

Layout of the pathogenicity test.

<p>(a) A pathogen-free pre-germinated lima bean was placed on a layer of sterilized, moist sea sand in a 50-ml sterile test tube. A layer (1 cm) of sand was added, and a piece of PDA containing hyphae of the fungus was placed on the sand. The tube was filled with sand up to 2 cm from the top, closed loosely with a cap, and incubated at 20°C under dim light in a growth room. (b) Seedlings reached a height of 10–12 cm (unless heavily damaged) and were observed for symptoms at 20 dpi. Symptoms shown in (b) were caused by <i>Macrophomina phaseolina</i>.</p

Types of symptoms in beans sampled from storehouses and grown in sterilized soil under controlled conditions.

<p>Plants were inspected 15 days after planting. 1, Early death of roots and the seedling; 2, root rot and poor emergence of the seedling; 3, necrosis in roots and poor growth of the seedling; 4, discoloration (mild necrosis) at the neck of the tap root (indicated by the black arrow).</p

Detection of PvEV-1 by RT-PCR in common beans in Tanzania.

<p>In the list below, landraces are marked with asterisk (*). Other samples represent improved varieties (origin of samples shown in parenthesis). Lane labelled ‘M’ represents a O'GeneRuler 1 kb Plus DNA ladder. The expected size of PCR products was 374 bp. Lanes 1, ‘Njugu’* (Southern Highlands zone); 2, ‘pooled RNA’ (Southern Highlands zone); 3, ‘pooled RNA’ (Eastern zone); 4, ‘pooled RNA’ (Northern zone); 5, ‘Rosekoko’/’Lyamungu 85’ (Eastern zone); 6, ‘Salundi’ (Southern Highlands zone); 7, ‘E 36’ (Southern Highlands zone); 8, ‘Msafiri’* (Southern Highlands zone); 9, ‘Msafiri’* (Eastern zone); and 10, ‘Mshindi’ (Eastern zone).</p