Pioneer plant species and fungal root endophytes in metal-polluted tailings deposited near human populations and agricultural areas in Northern Mexico.

As a consequence of industrial mining activity, high volumes of tailings are scattered around Mexico. Frequently, tailings contain heavy metals (HM) which entail threats against all organisms. The aim of this research was to identify plants and root fungal endophytes in polymetallic polluted tailings with the potential to be used in strategies of bioremediation. Four deposits of mine wastes, situated in a semi-arid region near urban and semi-urban populations, and agricultural areas, were studied. The physical and chemical characteristics of substrates, accumulation of HM in plant tissues, root colonization between arbuscular mycorrizal (AMF) and dark septate endophyte (DSE) fungi, and the identification of DSE fungi isolated from the roots of two plant species were studied. Substrates from all four sites exhibited extreme conditions: high levels in sand; low water retention; poor levels in available phosphorus and nitrogen content; and potentially toxic levels of lead (Pb), cadmium (Cd), and zinc (Zn). The native plants Lupinus campestris, Tagetes lunulata, and Cerdia congestiflora, as well as the exotic Cortaderia selloana and Asphodelus fistulosus, demonstrated a relevant potential role in the phytostabilization and/or phytoextraction of Pb, Cd, and Zn, according to the accumulation of metal in roots and translocation to shoots. Roots of eleven analyzed plant species were differentially co-colonized between AMF and DSE fungi; the presence of arbuscules and microsclerotia suggested an active physiological interaction. Fourteen DSE fungi were isolated from the inner area of roots of T. lunulata and Pennisetum villosum; molecular identification revealed the predominance of Alternaria and other Pleosporales. The use of native DSE fungi could reinforce the establishment of plants for biological reclamation of mine waste in semi-arid climate. Efforts are needed in order to accelerate a vegetation practice of mine wastes under study, which can reduce, in turn, their potential ecotoxicological impact on organisms, human populations, and agricultural areas.

Virulence testing and extracellular subtilisin-like (Pr1) and trypsin-like (Pr2) activity during propagule production of Paecilomyces fumosoroseus isolates from whiteflies (Homoptera: Aleyrodidae).

To properly characterize several isolates of Paecilomyces fumosoroseus, a fungal entomopathogen of whiteflies (Homoptera: Aleyrodidae) and other insect pests for biocontrol purposes, virulence towards Trialeurodes vaporariorum, and subtilisin-like (Pr1) and trypsin-like (Pr2) protease activity during propagule production were investigated in monospore cultures (MCs). The virulence of three MCs towards second instar whiteflies was measured and expressed as lethal median concentration (LC50). Number and widthlength ratio of propagules (blastospores, hyphal bodies, short hyphae, submerged conidia) and extracellular proteolytic activity was determined simultaneously in liquid medium. Total protease activity was assayed with azocasein, Pr1 and Pr2 activity was determined with the substrates N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-Benzoyl-Phe-Val-Arg-pnitroanilide, respectively. Natural variability in virulence, propagule production and cuticle-degrading proteases among isolates was observed. Bioassays showed a LC50 of 1.1 x 1,000, 2.5 x 10,000 and 7.6 x 10,000 conidia/ml for MCs EH-506/3, EH-503/3 and EH-520/3, respectively, EH-506/3 being the most virulent isolate. Isolate EH-503/3 produced the highest yield of propagules (7.7 x 10000000 propagules/ml), followed by EH-520/3 with 6.4 x 10000000 and EH-506/3 with 1.0 x 10000000 propagules/ml. Subtilisin-like (Pr1) and trypsin-like (Pr2) activity was present in the three MCs. Subtilisin-like (Pr1) activity was highest (745.7 UPr1/ml at 120 h) in the most virulent isolate, EH-506/3, pointing at Pr1 as a phenotypic marker of virulence for P. fumosoroseus. EH-506/3 appears to be a good candidate for whitefly biocontrol due to its high virulence, Pr1 concentration and rapid transition to blastospores in submerged liquid medium.

Virulence testing and extracellular subtilisin-like (Pr1) and trypsin-like (Pr2) activity during propagule production of Paecilomyces fumosoroseus isolates from whiteflies (Homoptera: Aleyrodidae)

Con el objetivo de caracterizar aislamientos para el control biológico demosquita blanca (Homoptera: Aleyrodidae), se estudiaron tres cultivosmonospóricos (CMs) de Paecilomyces fumosoroseus, hongo entomopatógenode la mosquita blanca (Homoptera: Aleyrodidae). Se determinó la virulencia enninfas de segundo estadio de la mosquita blanca expresada comoconcentración letal media (CL50). Se determinó la producción de propágulosfúngicos (blastosporas, cuerpos hifales, hifas cortas, conidios sumergidos)en medio líquido, se midió la relación de largo y ancho de los propágulos,y la actividad proteolítica (total; tipo subtilisina, Pr1 y tipo tripsina, Pr2)simultáneamente con la producción de propágulos. La proteasa total sedeterminó con azocaseina, y las actividades de Pr1 y Pr2 con N-Succinil-Ala-Ala-Pro-Phe-p-nitroanilida y N-Benzoil-Phe-Val-Arg-p-nitroanilida comosustratos específicos, respectivamente. Se observó variabilidad entre los tresCMs en cuanto a la virulencia, producción de propágulos y proteasas. Losbioensayos mostraron una CL50 de 1,1 x 103, 2,5 x 104 y 7,6 x 104 conidios/mlpara los CMs EH-506/3, EH-503/3 y EH-520/3, respectivamente.El CM donde se observó el mayor número de propágulos en las condicionesensayadas fue EH-503/3 (7,7 x 107), luego EH-520/3 con 6,4 x 107 y EH-506/3con 1,0 x 107 propágulos/ml. Las actividades enzimáticas de Pr1 y Pr2 sedemostraron en los tres CMs. La actividad tipo subtilisina (Pr1) fue mayor enel aislado más virulento (EH-506/3) con 745,7 UPr1/ml a las 120 h, y señala aPr1 como un marcador fenotípico de virulencia para P. fumosoroseus.EH-506/3 es un buen candidato para el control biológico de la mosquitablanca en México, por su alta virulencia, elevada concentración de Pr1 y surápida transición a blastosporas en el medio líquido ensayado(AU)

To properly characterize several isolates of Paecilomyces fumosoroseus,a fungal entomopathogen of whiteflies (Homoptera: Aleyrodidae) and otherinsect pests for biocontrol purposes, virulence towards Trialeurodesvaporariorum, and subtilisin-like (Pr1) and trypsin-like (Pr2) protease activityduring propagule production were investigated in monospore cultures (MCs).The virulence of three MCs towards second instar whiteflies was measuredand expressed as lethal median concentration (LC50). Number andwidth–length ratio of propagules (blastospores, hyphal bodies, short hyphae,submerged conidia) and extracellular proteolytic activity was determinedsimultaneously in liquid medium. Total protease activity was assayed withazocasein, Pr1 and Pr2 activity was determined with the substratesN-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-Benzoyl-Phe-Val-Arg-pnitroanilide,respectively. Natural variability in virulence, propagule productionand cuticle-degrading proteases among isolates was observed. Bioassaysshowed a LC50 of 1.1 x 103, 2.5 x 104 and 7.6 x 104 conidia/ml for MCsEH-506/3, EH-503/3 and EH-520/3, respectively, EH-506/3 being the mostvirulent isolate. Isolate EH-503/3 produced the highest yield of propagules(7.7 x 107 propagules/ml), followed by EH-520/3 with 6.4 x 107 and EH-506/3with 1.0 x 107 propagules/ml. Subtilisin-like (Pr1) and trypsin-like (Pr2) activitywas present in the three MCs. Subtilisin-like (Pr1) activity was highest(745.7 UPr1/ml at 120 h) in the most virulent isolate, EH-506/3, pointing at Pr1as a phenotypic marker of virulence for P. fumosoroseus. EH-506/3 appears tobe a good candidate for whitefly biocontrol due to its high virulence,Pr1 concentration and rapid transition to blastospores in submerged liquidmedium(AU)