A novel proof of concept for capturing the diversity of endophytic fungi preserved in herbarium specimens

Herbarium specimens represent important records of morphological and genetic diversity of plants that inform questions relevant to global change, including species distributions, phenology and functional traits. It is increasingly appreciated that plant microbiomes can influence these aspects of plant biology, but little is known regarding the historic distribution of microbes associated with plants collected in the pre-molecular age. If microbiomes can be observed reliably in herbarium specimens, researchers will gain a new lens with which to examine microbial ecology, evolution, species interactions. Here, we describe a method for accessing historical plant microbiomes from preserved herbarium specimens, providing a proof of concept using two plant taxa from the imperiled boreal biome ( Andromeda polifolia and Ledum palustre subsp . groenlandicum, Ericaceae). We focus on fungal endophytes, which occur within symptomless plant tissues such as leaves. Through a three-part approach (i.e. culturing, cloning and next-generation amplicon sequencing via the Illumina MiSeq platform, with extensive controls), we examined endophyte communities in dried, pressed leaves that had been processed as regular herbarium specimens and stored at room temperature in a herbarium for four years . We retrieved only one endophyte in culture, but cloning and especially the MiSeq analysis revealed a rich community of foliar endophytes. The phylogenetic distribution and diversity of endophyte assemblages, especially among the Ascomycota, resemble endophyte communities from fresh plants collected in the boreal biome. We could distinguish communities of endophytes in each plant species and differentiate likely endophytes from fungi that could be surface contaminants. Taxa found by cloning were observed in the larger MiSeq dataset, but species richness was greater when subsets of the same tissues were evaluated with the MiSeq approach. Our findings provide a proof of concept for capturing endophyte DNA from herbarium specimens, supporting the importance of herbarium records as roadmaps for understanding the dynamics of plant-associated microbial biodiversity in the Anthropocene. This article is part of the theme issue ‘Biological collections for understanding biodiversity in the Anthropocene’.

First Report of Actinidia virus A and Actinidia virus B on Kiwifruit in China

At present, two viruses affecting kiwifruit (Actinidia spp.), Actinidia virus A (AcVA) and Actinidia virus B (AcVB), both belonging to the genus Vitivirus in the family Betaflexiviridae, have been reported from New Zealand (2). The infected trees showed leaf vein chlorosis, flecking, and ringspots. China is the largest commercial kiwifruit producer. During field investigations in the growing season of 2013, symptoms of leaf chlorosis or ringspots, similar to those caused by AcVA and AcVB (1), were observed on some kiwifruit (Actinidia chinensis) plants in Hubei Province in the central China. Leaf samples were collected from three symptomatic and two symptomless plants of two A. chinensis cultivars. Total nucleic acids were extracted from the samples using a CTAB-based protocol described by Li et al. (3) and used as template in RT-PCR for the detection of AcVA and AcVB. Each virus was detected using two sets of primers reported by Blouin et al. (1). Primer sets AcVA 1F/1R and AcVA5F/5R were used for the AcVA detection, and AcVB1F/1R and AcVB5F/Viti3'R were used for the AcVB detection. AcVA was detected in three symptomatic plants (ID: Ac-HN-1, Ac-HN-3, and Ac-HN-5), and AcVB was detected in two symptomatic plants (ID: Ac-HN-1 and Ac-HN-3) and in one symptomless plant (ID: Ac-HN-2). Neither virus was detected in the second symptomless plant (ID: Ac-HN-4). Samples Ac-HN-1 and Ac-HN-3 had mixed infection of AcVA and AcVB, and sample Ac-HN-2 had the latent infection of AcVB. The sequenced 283-bp RT-PCR amplicons of the replicase-encoding gene from AcVA isolates AC-HN-3 and AC-HN-5 using AcVA1F/1R shared 90.8% nucleotide (nt) identity with the corresponding sequence of the New Zealand AcVA isolate (GenBank Accession No. JN427014.1). The 269-bp fragments of the RNA-binding protein-encoding gene obtained by using AcVA5F/5R shared 85.5 to 85.9% nt identities with the corresponding sequence of JN427014.1. The AcVB5F/Viti3'R products of 365 to 369 bp from three AcVB isolates shared 85.5 to 88.6% nt identities with the corresponding sequence of the New Zealand AcVB isolate. The representative sequences were submitted to GenBank with accession numbers KJ696776 and KJ696777 for the 269-bp fragments of AcVA-HN-1 and AcVA-HN-3, and KJ696778 and KJ696779 for the 365-bp and 369-bp fragments of AcVB-HN-1 and AcVB-HN-2, respectively. In addition, 12 and 14 out of 42 kiwi samples (excluding HN-1 to HN-5) collected randomly were positive for AcVA and AcVB as detected by RT-PCR. Meanwhile, the sample affected by AcVA-HN-5 was subjected to deep sequencing of the small RNAs (sRNAs) for complete survey of the infecting viruses. De novo assembly of sRNAs generated four sequence contigs, with lengths ranging from 161 to 285 nt, matching to ORFs 1 to 3 of the genome of the New Zealand AcVA isolate with significant nucleotide (91 to 95%) and amino acid (80 to 94%) similarities, and some other contigs from a new virus (unpublished). The result further confirmed AcVA infection in the kiwi plant. To our knowledge, this is the first report of both AcVA and AcVB outside of New Zealand. The Chinese isolates of the two viruses are distinct from those reported from New Zealand. The results provide valuable information for improving the viral sanitary status of the kiwifruit germplasm in China. References: (1) A. G. Blouin et al. Arch. Virol. 157:713, 2012. (2) A. G. Blouin et al. J. Plant Pathol. 95:221, 2013. (3) R. Li et al. J. Virol. Methods 154:48, 2008.

First Report of ‘Candidatus Phytoplasma trifolii’-Related Strain Associated with Safflower Phyllody Disease in Iran

During a survey in 2003, safflower plants (Carthamus tinctorius) with phyllody symptoms were observed in production fields in several districts of Fars and Yazd provinces in Iran. Affected plants showed floral virescence, phyllody, proliferation of axillary buds, and little leaf symptoms. Incidence of the disease was less than 10%. Direct and nested PCR assays were used to verify association of phytoplasma with the disease. Total DNA was extracted from fresh, fine roots of eight phyllody-affected safflower plants and one symptomless plant. With phytoplasma universal primer pair P1/P7 (5′-AAGAGTTTGATCCTGGCTCAGGATT-3′/5′-CGTCCTTCATCGGCTCTT-3′), target DNA fragments of approximately 1.8 kb were amplified by direct PCR from phyllody-affected plants and Iranian cabbage yellows (ICY) phytoplasma used as a positive control. Reamplification of P1/P7 products with 16S rRNA gene primer pair R16F2n/R16R2 (5′-GAAACGACTGCTAAGACTGG-3′/5′-TGACGGGCGGTGTGTACAAACCCCG-3′) yielded fragments of the expected size (1.2 kb) from all eight diseased plants and the ICY-positive control. No products were amplified from the symptomless plant by either assay. R16F2n/R16R2 products were subjected to restriction fragment length polymorphism (RFLP) analysis by separate digestion with AluI, HaeIII, HhaI, HinfI, HpaII, MseI, RsaI, Sau3AI, or TaqI endonuclease. Comparison of resulting RFLP patterns with published patterns of other phytoplasmas (2) tentatively identified safflower phyllody (SP) phytoplasma as a member of clover proliferation group 16SrVI, subgroup C. HhaI digests also differentiated SP from ICY phytoplasma, a previously reported subgroup 16SrVI-A strain (3). After sequencing of the 16S rDNA fragment (GenBank Accession No. DQ88948), a BLAST search determined that SP phytoplasma shared closest homology with 16SrVI group members (‘Candidatus Phytoplasma trifolii’) and related strains (4). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SP phytoplasma to be most similar (99.7%) to brinjal little leaf (BLL) phytoplasma (GenBank Accession No. X83431). Analysis of putative restriction sites in 16S rRNA gene sequences revealed that SP and BLL shared identical restriction profiles and that both differed from the ‘Ca. Phytoplasma trifolii’ reference strain (GenBank Accession No. AY390261) because of the absence of a single HhaI site and the presence of an additional MseI site. Although safflower phyllody disease has been previously reported in Israel, the associated phytoplasma was classified as a strain of the aster yellows subgroup 16SrI-B (1). To our knowledge, this is the first report of safflower as a host of a ‘Ca. Phytoplasma trifolii’-related strain. References: (1) M. Klein. Plant Dis. Rep. 54:735, 1970. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (3) M. Salehi et al. Plant Dis. 91:625, 2007. (4) K. Wang and C. Hiruki, Phytopathology 91:546, 2001.

Detection of Colletotrichum acutatum latent infections in strawberry petioles and leaves

Colletotrichum acutatum is the most significant agent of anthracnose strawberry fruit rot. Besides being a necrotrophic pest, it can spend a part of its life cycle as an epiphyte, in a form of latent infection. The presence of the fungi on symptomless plant tissue is considered one of the main ways of distribution of this economically harmful pathogen in the world. Investigation of latent C. acutatum infection was carried out on artificially inoculated strawberries. The initiation of fungi sporulation on symptomless petioles and leaves was carried out by exposing them to the herbicide paraquat (0.25%) and low temperatures, which caused plant tissue decay in different ways. Surface sterilization with 0.5% NaOCl precedes the exposure of plant material to paraquat. The freezing procedure was carried out by exposure of plant material to the temperature of -20?C for 2h. After the freezing, one group was rinsed in Tween 20 (18 ?l/l), and another group underwent surface sterilization in 0.0525% NaOCl with an addition of Tween 20 (18 ?l/l). After 6 days of incubation, the appearance of acervuli and conidia was detected in 93.33 to 100% plant parts exposed to paraquat treatment and freezing procedure. In inoculated parts which were not exposed to herbicides or low temperatures, the presence of acervuli was detected in 3.33% tested petioles and 6.67% leaves.

First Report of Cucurbit yellow stunting disorder virus in Morocco

In October, 1999, severe yellowing symptoms were observed in a melon (Cucumis melo L.) crop grown under plastic tunnels in the region of Agadir, Morocco. Large populations of whiteflies (Bemisia tabaci) were noticed during the early stages of the crop. At harvest, leaf samples were collected from two symptomatic plants and one symptomless plant. A mature yellow leaf was assayed from each symptomatic plant and for one of these two plants a younger leaf exhibiting only yellow spots. Cucurbit aphid-borne yellows virus, which causes similar symptoms in melons, was not detected by double-antibody sandwich enzyme-linked immunosorbent assay tests. Total RNA was extracted from fresh leaf tissues and submitted to reverse transcription and polymerase chain reaction with primers specific to two whitefly-transmissible viruses: Beet pseudo-yellows virus (BPYV) and Cucurbit yellow stunting disorder virus (CYSDV) (2). No amplification was obtained with BPYV-specific primers. In contrast, an expected 465-bp product was amplified in all samples from symptomatic plants with CYSDV-specific primers. No amplification was detected in samples from the symptomless plant nor from healthy control plants. B. tabaci-transmitted CYSDV has been reported in the Middle East, southwestern Europe, and North America (1,4). This is the first report of CYSDV in Morocco, and it follows the first report of another B. tabaci-transmitted virus, Tomato yellow leaf curl virus, in tomato (3), suggesting an important change in the viral pathosystem affecting vegetable crops in Morocco. References: (1) Kao et al. Plant Dis. 84:101, 2000. (2) Livieratos et al. Plant Pathol. 47:362, 1998. (3) Peterschmitt et al. Plant Dis. 83:1074, 1999. (4) Wisler et al. Plant Dis. 82:270, 1998.

Resistance to Bean Golden Mosaic Virus in Bean Genotypes

Bean lines MD 806, MD 807, MD 820, MD 829, MD 808, and MD 821, all with high levels of resistance to bean golden mosaic virus (BGMV), were selected under field conditions. On a 1 to 9 scale (1 = symptomless plant; 9 = severe symptoms), the mean disease severity of the MD lines ranged from 1.7 to 3.9 for mosaic symptom (foliar yellowing) and from 1.7 to 2.2 for malformation. In contrast, disease severity in the susceptible cultivars Carioca, Carnaval, and Iapar 20 ranged from 4.1 to 8.7 for mosaic and from 4.0 to 7.3 for malformation. Symptoms on the selected lines were expressed later than on susceptible controls. The MD lines yielded 1.2- to 5.9-fold the yield of the cultivars Carioca and Iapar 20. Under greenhouse conditions, reactions of the bean genotypes to BGMV were similar to those observed in the field. MD 806 and MD 807 were obtained from early crosses involving genotypes that reacted to BGMV infection with mild mosaic (Aeté 1/38 and TMD-1) and mild malformation (Porrillo Sintético and Turrialba 1). Parents of the lines MD 820 and MD 829 were Phaseolus coccineus (of unknown reaction to BGMV) and Carioca. Line MD 648 (DOR-selected line) was the ancestral parent of lines MD 808 and MD 821, which had the highest levels of resistance or tolerance to BGMV. Severity of all BGMV symptoms showed gradual reductions from the earliest parents to their later selected progenies. Responses of the parent genotypes and progenies to BGMV infection suggest a combination of resistance characters in the selected MD lines. MD 806, MD 820, and MD 821 were released in the state of Paraná under the names Iapar 57, Iapar 72, and Iapar 65, respectively.