Indirect interactions among co-infecting parasites and a microbial mutualist impact disease progression

Interactions among parasites and other microbes within hosts can impact disease progression, yet study of such interactions has been mostly limited to pairwise combinations of microbes. Given the diversity of microbes within hosts, indirect interactions among more than two microbial species may also impact disease. To test this hypothesis, we performed inoculation experiments that investigated interactions among two fungal parasites, Rhizoctonia solani and Colletotrichum cereale, and a systemic fungal endophyte, Epichloë coenophiala, within the grass, tall fescue ( Lolium arundinaceum ). Both direct and indirect interactions impacted disease progression. While the endophyte did not directly influence R. solani disease progression or C. cereale symptom development, the endophyte modified the interaction between the two parasites . The magnitude of the facilitative effect of C. cereale on the growth of R. solani tended to be greater when the endophyte was present. Moreover, this interaction modification strongly affected leaf mortality. For plants lacking the endophyte, parasite co-inoculation did not increase leaf mortality compared to single-parasite inoculations. By contrast, for endophyte-infected plants, parasite co-inoculation increased leaf mortality compared to inoculation with R. solani or C. cereale alone by 1.9 or 4.9 times, respectively. Together, these results show that disease progression can be strongly impacted by indirect interactions among microbial symbionts.

Higher-order interactions among coinfecting parasites and a microbial mutualist impact disease progression

Interactions among parasites and other microbes within hosts can impact disease progression, yet study of such interactions has been mostly limited to pairwise combinations of microbes. Given the diversity of microbes within hosts, higher-order interactions among more than two microbial species may also impact disease. To test this hypothesis, we performed inoculation experiments that investigated interactions among two fungal parasites, Rhizoctonia solani and Colletotrichum cereale, and a systemic fungal endophyte, Epichloê coenophiala, within a grass host. Both pairwise and higher-order interactions impacted disease progression. While the endophyte did not directly influence R. solani growth or C. cereale symptom development, the endophyte modified the interaction between the two parasites. The magnitude of the facilitative effect of C. cereale on the growth of R. solani tended to be greater when the endophyte was present. Moreover, this interaction modification strongly affected leaf mortality. For plants lacking the endophyte, parasite co-inoculation did not increase leaf mortality compared to single-parasite inoculations. In contrast, for endophyte-infected plants, parasite co-inoculation increased leaf mortality compared to inoculation with R. solani or C. cereale alone by 1.9 or 4.9 times, respectively. Together, these results show that disease progression can be strongly impacted by higher-order interactions among microbial symbionts.

First Report of Colletotrichum cereale Causing Anthracnose on Avena nuda in China

Naked oats (Avena nuda L.) is rich in protein, fat, vitamin, mineral elements and so on, and is one of the world's recognized cereal crops with the highest nutritional and healthcare value. In July 2019, leaf spot was detected on A. nuda in Zhangbei experimental station of Hebei Agricultural University. The incidence of disease is 10% to 20%. The symptoms were similar to anthracnose disease, the infected leaves had fusiform or nearly fusiform yellowish-brown spots, yellow halo around the spots. Numerous acervuli with black setae diagnostic of fungi in the genus Colletotrichum were present on necrotic lesions. To identify the pathogen, ten symptomatic leaves were collected, and only one disease spot was isolated from each leaf. Small square leaf pieces (3 to 5 mm) were excised from the junction of diseased and healthy tissues with a sterile scalpel and surface disinfested with 75% alcohol for 30s, 0.1% corrosive sublimate for 1 min, rinsed three times in sterile water. Plant tissues were then transferred on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Two fungal isolates were obtained and purified by single-spore isolation method. All fungi have the same morphology and no other fungi were isolated. The aerial mycelium was gray black. The conidia were colorless and transparent, falcate, slightly curved, tapered toward the tips, and produced in acervuli with brown setae. The length and width of 100 conidia were measured and size ranged from 1.86 to 3.84 × 8.62 to 29.81 μm. These morphological characteristics were consistent with the description of Colletotrichum cereale (Crouch et al. 2006). To further assess the identity of the species, the genomic DNA of two fungal isolates (LYM19-4 and LYM19-10) was extracted by a CTAB protocol. The ribosomal DNA internal transcribed spacer (ITS) region as well as, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and the beta-tubulin 2 (Tub2) partial genes were amplified and sequenced with primers ITS4/5, GDF/GDR, ACT-512F/ACT-783R, and T1/Bt2b, respectively (Carbone et al. 1999; Templeton et al. 1992; O'Donnell et al. 1997; Glass et al. 1995). The sequences of the ITS-rDNA region (MW040121, MW040122), the GAPDH sequences (MW052554, MW052555), the ACT sequences (MW052556, MW052551) and the Tub2 sequences (MW052552, MW052553) of the two single-spore isolates were more than 99% identical to C. cereale isolate CGMCC3.15110 (JX625159, KC843517, KC843534 and JX625186). Maximum likelihood tree based on concatenated sequences of the four genes were constructed using MEGA7. The results showed the strains isolated from A. nuda were closely related to C. cereale, as supported by high bootstrap values. A pathogenicity test of the C. cereale isolates was performed on first unfolding leaves of A. nuda. Koch's postulates were carried out with isolates by spraying a conidial suspension of 106 conidia/mL on leaves of healthy A. nuda. Four replicated pots were inoculated at a time, 10 leaves each pot, while sterile distilled water was used as the control. All treated plants were placed in a moist chamber (25°C, 16-h light and 8-h dark period). Anthracnose symptoms developed on the inoculated plants 7 days post inoculation while all control plants remained healthy. Microscopic examination showed the surface of infected leaves had the same acervuli, setae, and conidia as the original isolate. The pathogenicity test was repeated three times. C. cereale was previously reported as the causal agent of anthracnose on feather reed grass in US (Crouch et al. 2009). To our knowledge, this is the first report of C. cereale as the causal agent of A. nuda anthracnose in China.

Sand Size Affects Topdressing Removed by Mowing and Anthracnose on Annual Bluegrass Putting Green Turf

Sand size can affect the ability to incorporate topdressing into the turf canopy and thatch on golf course putting greens; unincorporated sand interferes with mowing and play. This 3-year field trial was initiated to determine the effects of sand size on sand incorporation, surface wetness, and anthracnose (caused by Colletotrichum cereale Manns sensu lato Crouch, Clarke, and Hillman) of annual bluegrass [Poa annua L. f. reptans (Hausskn) T. Koyama] maintained as a putting green. The experimental design was a randomized complete block with four replications; treatments included a non-topdressed control and three topdressing sands (medium-coarse, medium, or medium-fine) applied every 2 weeks at 0.15 L·m−2 during the summer. Topdressing with medium-coarse sand was more difficult to incorporate than the medium and medium-fine sands, resulting in a greater quantity of sand collected with mower clippings. Analyzing the particle distribution of sand removed by mowing confirmed that coarser sand particles were more likely to be removed in mower clippings. Surface wetness measured as volumetric water content (VWC) at the 0- to 38-mm depth zone was greater in non-topdressed plots than topdressed plots on 35% of observations. Few differences in VWC were found among sand size treatments. Turf responses to topdressing were not immediate; however, as sand accumulated in the turf canopy, topdressed plots typically had lower anthracnose severity than non-topdressed turf after the first year. Additionally, topdressing with medium and medium-fine sands produced similar or occasionally lower disease severity than topdressing with medium-coarse sand. The lack of negative effects of medium and medium-fine sands combined with better incorporation after topdressing and less disruption to the putting surface should allow golf course superintendents to apply topdressing at frequencies and/or quantities needed during the summer to maintain high-quality turf and playing conditions.

Anthracnose Disease on Annual Bluegrass as Affected by Foot Traffic and Sand Topdressing

Sand topdressing is applied to maintain or enhance playability of the turf surface of putting greens. Anthracnose is a devastating disease of annual bluegrass (ABG; Poa annua) putting green turf, caused by Colletotrichum cereale. The disease is more severe on weakened turf and reputed to be exacerbated by management practices that wound turf. A 2-year field study was initiated in 2007 to evaluate the effects of foot traffic (0 versus 327 footsteps m−2, equivalent to 200 rounds day−1) and sand topdressing (0 and 0.3 liter m−2 every week) on anthracnose severity of ABG mowed at 3.2 mm. Surprisingly, foot traffic reduced anthracnose severity as much as 28%, regardless of sand topdressing, during both years. Although sand topdressing initially increased disease severity (up to 7%) in 2007, continued applications decreased severity by 9% later in August 2007 and again in 2008. The treatment combination of foot traffic 5 days week−1 and weekly sand topdressing resulted in the best turf quality by the end of both seasons. Results indicate that the practice of sand topdressing can be continued even under conditions of intense foot traffic and anthracnose disease development on ABG putting greens.

Identification and Molecular Mapping of a Wheat Gene for Resistance to an Unadapted Isolate of Colletotrichum cereale

To elucidate genetic mechanisms of host species specificity between graminicolous anthracnose fungi and gramineous plants, infection assays were performed with a Sorghum isolate (Colletotrichum sublineolum), a Digitaria isolate (C. hanaui), a Polypogon isolate (C. cereale), and an Avena isolate (C. cereale). They were specifically virulent on the plants from which they were isolated. When 72 wheat lines were inoculated with an unadapted isolate from Asia Minor bluegrass (Cgp29), however, some exceptional cultivars were recognized. Although most cultivars were resistant to Cgp29, ‘Hope’ was susceptible. In F2 populations derived from crosses between three resistant cultivars—‘Norin 4’ (N4), ‘Chinese Spring’ (CS), and ‘Shin-chunaga’ (Sch)—and the susceptible Hope, resistant and susceptible seedlings segregated in a 3:1 ratio, suggesting that a major gene is involved in the resistance of each cultivar to Cgp29. In F2 populations derived from crosses between the three resistant cultivars, all seedlings were resistant, suggesting that these three cultivars carry the same gene. This resistance gene was designated as “resistance to Colletotrichum cereale 1” (Rcc1). Analysis with the CS–Hope chromosome substitution lines and molecular mapping revealed that Rcc1 was located on the long arm of chromosome 5A. Cytologically, Rcc1 was mainly associated with hypersensitive reaction. These results suggest that major genes similar to those controlling cultivar specificity are involved in the resistance of wheat against the unadapted isolate of C. cereale.